From 290b2f47a8432736365591b79a7127e910130e76 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Mon, 15 Nov 2021 14:43:08 -0500 Subject: [PATCH 01/25] Add first stab at gating --- vignettes/project2_chunks/project2_tim.Rmd | 29 ++++++++++++++++++++++ 1 file changed, 29 insertions(+) diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 2f83a68..760f1c2 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -138,6 +138,35 @@ fracByCell <- do.call(cbind, smoothScatter(fracByCell) ``` +Suppose we want to implement some logic for gating cells. For example, we might say "if a cell has greater than 0.03 human transcriptomes expressed, less than 0.2 human transcriptomes, and less than 0.2 mouse transcriptomes, we'll call it human". Needless to say this is a bit of a craft. Let's implement it. + +```{r, gating} + +# could do this "tidy" style on a tibble +is_human <- function(human, mouse, minHuman=0.03, maxHuman=0.2, maxMouse=0.03) { + if (mouse > maxMouse) return(FALSE) + if (human > minHuman & human < maxHuman) return(TRUE) + else return(FALSE) +} + +# note redudancy +is_mouse <- function(human, mouse, minMouse=0.03, maxMouse=0.2, maxHuman=0.03) { + if (human > maxHuman) return(FALSE) +  if (mouse > minMouse & mouse < maxMouse) return(TRUE) +  else return(FALSE) +} + +classified <- function(human, mouse) { + if (is_mouse(human=human, mouse=mouse) & is_human(human=human, mouse=mouse)) return(FALSE) + if (is_mouse(human=human, mouse=mouse) | is_human(human=human, mouse=mouse)) return(TRUE) + else return(FALSE) +} + +fractibble <- tibble(fracByCell) +fractibble %>% mutate(classifiable = classified(human, mouse)) -> classified + +``` + _Question: Can you do this again but for each method?_ _Question: Can you do this again but for each mixture?_ From 650d743a9eefea96a969880f81972dc9a7d133c6 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Mon, 15 Nov 2021 14:49:01 -0500 Subject: [PATCH 02/25] Update project2_tim.Rmd Add gating logic (attempting to do so tidily, may have to resort to apply()) --- vignettes/project2_chunks/project2_tim.Rmd | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 760f1c2..0aecc97 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -162,7 +162,7 @@ classified <- function(human, mouse) { else return(FALSE) } -fractibble <- tibble(fracByCell) +fractibble <- tibble(data.frame(fracByCell)) fractibble %>% mutate(classifiable = classified(human, mouse)) -> classified ``` From efed9284b30063dd69e071db5ba323601e6cd735 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Mon, 15 Nov 2021 14:55:07 -0500 Subject: [PATCH 03/25] Update project2_tim.Rmd just use apply . --- vignettes/project2_chunks/project2_tim.Rmd | 19 ++++++++++--------- 1 file changed, 10 insertions(+), 9 deletions(-) diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 0aecc97..dda25fb 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -143,26 +143,27 @@ Suppose we want to implement some logic for gating cells. For example, we might ```{r, gating} # could do this "tidy" style on a tibble -is_human <- function(human, mouse, minHuman=0.03, maxHuman=0.2, maxMouse=0.03) { - if (mouse > maxMouse) return(FALSE) - if (human > minHuman & human < maxHuman) return(TRUE) +is_human <- function(x, minHuman=0.03, maxHuman=0.2, maxMouse=0.03) { + if (x['mouse'] > maxMouse) return(FALSE) + else if (x['human'] > minHuman & x['human'] < maxHuman) return(TRUE) else return(FALSE) } # note redudancy -is_mouse <- function(human, mouse, minMouse=0.03, maxMouse=0.2, maxHuman=0.03) { +is_mouse <- function(x, mouse, minMouse=0.03, maxMouse=0.2, maxHuman=0.03) { if (human > maxHuman) return(FALSE) -  if (mouse > minMouse & mouse < maxMouse) return(TRUE) +  else if (mouse > minMouse & mouse < maxMouse) return(TRUE)  else return(FALSE) } -classified <- function(human, mouse) { - if (is_mouse(human=human, mouse=mouse) & is_human(human=human, mouse=mouse)) return(FALSE) - if (is_mouse(human=human, mouse=mouse) | is_human(human=human, mouse=mouse)) return(TRUE) +classified <- function(x) { + if (is_mouse(x) & is_human(x)) return(FALSE) + else if (is_mouse(x) | is_human(x)) return(TRUE) else return(FALSE) } -fractibble <- tibble(data.frame(fracByCell)) +frac <- data.frame(fracByCell) +frac$classifiable <- apply(frac, 1, classified) fractibble %>% mutate(classifiable = classified(human, mouse)) -> classified ``` From b3e860214727ac62e0fc8096f3e91e311f8e8f0b Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Mon, 15 Nov 2021 14:57:21 -0500 Subject: [PATCH 04/25] Update project2_tim.Rmd typo --- vignettes/project2_chunks/project2_tim.Rmd | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index dda25fb..9b1c689 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -150,9 +150,9 @@ is_human <- function(x, minHuman=0.03, maxHuman=0.2, maxMouse=0.03) { } # note redudancy -is_mouse <- function(x, mouse, minMouse=0.03, maxMouse=0.2, maxHuman=0.03) { - if (human > maxHuman) return(FALSE) -  else if (mouse > minMouse & mouse < maxMouse) return(TRUE) +is_mouse <- function(x, minMouse=0.03, maxMouse=0.2, maxHuman=0.03) { + if (x['human'] > maxHuman) return(FALSE) +  else if (x['mouse'] > minMouse & x['mouse'] < maxMouse) return(TRUE)  else return(FALSE) } @@ -164,7 +164,7 @@ classified <- function(x) { frac <- data.frame(fracByCell) frac$classifiable <- apply(frac, 1, classified) -fractibble %>% mutate(classifiable = classified(human, mouse)) -> classified + ``` From a482f1bfe43acbee4b34919b51e2e471ac7de48a Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 08:31:50 -0500 Subject: [PATCH 05/25] update vignette chunks --- DESCRIPTION | 10 +- vignettes/figure/SingleCellExperiment.png | Bin 0 -> 82024 bytes vignettes/figure/SummarizedExperiment.jpg | Bin 0 -> 57460 bytes vignettes/project2.Rmd | 4 +- vignettes/project2_chunks/project2_tim.Rmd | 482 ++++++++++++++++++--- 5 files changed, 438 insertions(+), 58 deletions(-) create mode 100644 vignettes/figure/SingleCellExperiment.png create mode 100644 vignettes/figure/SummarizedExperiment.jpg diff --git a/DESCRIPTION b/DESCRIPTION index fe15a42..238fb6e 100644 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -11,8 +11,8 @@ Description: For the practical sessions in Experimental Design & Biostats 2021, Rstudio to \url{https://rpubs.com/}, so all that's left is to make sure that dependencies for assignments are in place. This package also includes a few convenience functions (phelp, lsos, etc.). -Version: 0.11.15 -Date: 2021-11-15 +Version: 0.11.16 +Date: 2021-11-16 Authors@R: person("Triche", "Lab", role=c("aut", "cre"), email="trichelab@gmail.com") Author: Triche Lab [aut, cre] Maintainer: Triche Lab @@ -31,6 +31,7 @@ Imports: BiocManager, broom, broom.mixed, + coefplot, corrr, datasets, devtools, @@ -38,6 +39,7 @@ Imports: downlit, dplyr, factoextra, + forcats, ggforce, ggplot2, ggthemes, @@ -67,11 +69,15 @@ Imports: rpart, rpart.plot, rsample, + scater, scico, + SingleCellExperiment, skeletor, skimr, + SummarizedExperiment, tibble, tidyr, + tidySingleCellExperiment, tidytext, tidyverse, tune, diff --git a/vignettes/figure/SingleCellExperiment.png b/vignettes/figure/SingleCellExperiment.png new file mode 100644 index 0000000000000000000000000000000000000000..be022d1bf3bbad046015c710bcf6fa291d3be13c GIT binary patch literal 82024 zcmXVX1z40_*Yyx0DIpE=AR-`Lf`EWXcT0D7cM5`ph=52-Nh2v;Dkaig0@B^x-}e3g 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ziC5GyPp)rxSc#g`Q^_-jp~kqtfZ?D_d*A^A9w&$xG&;v`H#t8r+5kvYf%e3(?J74@ zwnchmQ7Id#x;MW2!RO%>1A*=xoS_tG_KIl5uE~toCXzWtFFS%1wCURdK%r_LOWB68 z;dst5#2#mim_oPK>=s?)W^@a*k1*`=J7D2xB2DY6ZPpDqkq;xHW`vB;b{<2>)`azh)heCN{FpW5`H% # stringr::str_split takes strings and a split pattern mutate(experiment = str_split(name, "\\.", simplify=TRUE)[,1]) %>% # column 1 mutate(method = str_split(name, "\\.", simplify=TRUE)[,2]) %>% # column 2 - mutate(cell = str_split(name, "\\.", simplify=TRUE)[,3]) -> # column 3 + mutate(barcode = str_split(name, "\\.", simplify=TRUE)[,3]) -> # column 3 celltibble # the result is assigned to the object `celltibble` # how many cells per mixture were run with each method? @@ -328,7 +328,7 @@ if (FALSE) { # if you want to recreate the SingleCellExperiment: } # if you just want to load it -tidybarnyard <- tidy(readRDS(url("https://ttriche.github.io/RDS/barnyard.rds"))) +tidybarnyard <- readRDS(url("https://ttriche.github.io/RDS/tidybarnyard.rds")) show(tidybarnyard) ``` diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 9b1c689..4e5165c 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -18,7 +18,7 @@ library(devtools) load_all("./") ``` -# Tim's stab at classification and plotting +# Background Recall that we are seeking to identify the influence of two factors on our ability to classify cells: the experimental vial and the library prep protocol. @@ -33,16 +33,7 @@ let us answer ANOVA-style questions: "how much variability is explained by a particular factor in the model?" First we need to set up our response variable, i.e., the ability to classify a cell confidently. For that, we need cutoffs. -![bus format](../figure/bustools.jpeg) - -The barcode, UMI, set or [BUS format](https://academic.oup.com/bioinformatics/article/35/21/4472/5487510) neatly encapsulates the process of transforming reads -resulting from a single-cell RNAseq experiment into counts of molecular barcodes -describing a cell, a molecule, and the likely genomic origin of that molecule. -It's one of the most compact representations possible for raw scRNAseq outputs, -and the figure shows how it facilitates direct comparisons. The Broad used their -`scumi` tool instead, but the basic notion ("make everything comparable") and -the resulting sparse matrix of counts is similar in spirit. - +# tidySingleCellExperiment With the release of Bioconductor 3.14, the project includes a tidy single cell experiment (data structure) package, which is great since all other single cell @@ -76,6 +67,7 @@ You could always adjust the `ideal` argument to the function below to resample: # adapted from a SingleCellExperiment-centric method for CITEseq sample_umis <- function(umis, meta, block, ideal=300) { + # {{{ stopifnot(nrow(meta) == ncol(umis)) stopifnot(length(block) == nrow(meta)) @@ -102,72 +94,454 @@ sample_umis <- function(umis, meta, block, ideal=300) { message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), " cells in ", length(samplesets), " blocks.") umis[, keep] + # }}} } - ``` -Load the data: +# Tim's stab at classification and plotting + +It will be difficult to evaluate our strategy if we don't first load the data: -```{r, createtidysce} +```{r, gettidysce } library(SingleCellExperiment) library(tidySingleCellExperiment) +# should package this... if (!exists("tidybarnyard")) tidybarnyard <- tidy(readRDS(url("https://ttriche.github.io/RDS/barnyard.rds"))) -rowGenome <- rowData(tidybarnyard)$genome + +``` +It will be useful to know that, when you use the `$` operator on a typical +Bioconductor object (such as a _tidySingleCellExperiment_ like ours), it assumes +you want to see the column data (`colData`) with that name (see below). For +example, when we built up the `tidybarnyard` object from the cell and gene +tables (by splitting and mutating them), we fed them to the SingleCellExperiment +constructor function and it bolted them onto the side of the object. It turns +out _[SingleCellExperiment](https://www.nature.com/articles/s41592-019-0654-x)_ +is just a modified version of _[SummarizedExperiment](https://www.nature.com/articles/nmeth.3252)_: + +![the SummarizedExperiment scheme](../figure/SummarizedExperiment.png) + +The figure from the original paper is better/easier to understand, although you +can certainly read about the new one. TidySingleCellExperiment wraps the "newer" +_[SingleCellExperiment](https://www.nature.com/articles/s41592-019-0654-x)_ +so that it plays somewhat more nicely with the tidyverse we've all been using. +There are many other approaches to analyzing single-cell data, and you may well +bump into several of them, but this one happens to be very efficient. Plus, +keeping sample- or cell-specific details lined up with columns, and gene- or +feature-specific details lined up with rows, tends to stay in style. The only +annoying feature (ha!) with this setup is that you need to explicitly tell R +when you want data about the rows themselves (i.e., the rowData). + +The _[tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html)_ package gives +us some tools to stay in the Tidyverse as much as possible while working with +single-cell data. (It turns out that the underlying data makes this somewhat +less trivial than you might expect. Fortunately, you don't have to care.) + +Concretely, let's see how these moving parts fit together. The `dim` function +provides dimensions for a rectangular object, rows x columns, which makes it +a little easier to keep track of what's going on when we subset either one. + +```{r, rowDataAndColumnData } + +# as what is our object masquerading? +show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" + +# how many rows (genes) and columns (cells) are there in our tidy barnyard? +dim(tidybarnyard) + +# is this equivalent to what we get from nrow() and ncol()? +identical(dim(tidybarnyard), + c(nrow(tidybarnyard), ncol(tidybarnyard))) + +# the assay(s) should also have this many rows and columns. Do they? +identical(dim(tidybarnyard), dim(assay(tidybarnyard))) + +# there is a shortcut for the `counts` assay, since it's so common: +counts(tidybarnyard)[1:3, 1:3] + +# does each row (gene) in the object have a corresponding rowData() row? +identical(rownames(tidybarnyard), rownames(rowData(tidybarnyard))) + +# does each column (cell) in the object have a corresponding colData() row? +identical(colnames(tidybarnyard), rownames(colData(tidybarnyard))) + +# is there a rowData row and a colData row that corresponds to each UMI count? +identical(dim(counts(tidybarnyard)), + c(nrow(rowData(tidybarnyard)), nrow(colData(tidybarnyard)))) + +# does that mean we can ask for a random cell with certain attributes? +as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" + filter(method == "inDrops") %>% # "choose rows where method == inDrops" + slice_sample %>% # "randomly slice out one row" + pull("cell") -> aCell # "pull the column cell, assign to aCell" + +# your cell: +show(aCell) + +# does that mean we can ask for a random gene with certain attributes? +as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" + filter(genome == "mm10") %>% # "choose rows where genome == mm10" + slice_sample %>% # "randomly slice out one row" + pull("name") -> aGene # "pull the column name, assign to aGene" + +# your gene: +show(aGene) + +# how many copies of this random gene were found in this random cell? +counts(tidybarnyard)[aGene, aCell] + +# note that the odds are good that you'll get a 0 for this random combination: +library(Matrix) # for the `nnzero` function +sparsity <- 1 - (nnzero(counts(tidybarnyard)) / # number of nonzero counts + prod(dim(tidybarnyard))) # number of rows * columns + +# specifically, the chance of getting a zero is about 92.3%: +message(round(sparsity * 100, 1), "%") + +# This is fairly typical for a single-cell experiment, perhaps a bit low even. +# You could also take 10000 or so random samples to estimate the sparsity. +# For example, you could grab 100 genes from 100 cells at a time: +as_tibble(tidybarnyard) %>% # turn tidybarnyard's colData into a tibble + slice_sample(n=100) %>% # slice out 100 random rows, and then... + pull("cell") -> aHundredCells # assign the "cell" column to "aHundredCells" + +as_tibble(rowData(tidybarnyard)) %>% # turn tidybarnyard's rowData into a tibble + slice_sample(n=100) %>% # slice out 100 random rows, and then + pull("name") -> aHundredGenes # assign "name" column to "aHundredGenes" + +samples <- (length(aHundredCells) * length(aHundredGenes)) +nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) +sparsity_hat <- (samples - nonzero) / samples +sparsity_hat # estimated sparsity + +# In fact, we can use this scheme to look at sampling error: +sample_sparsity <- function(object, cells=100, genes=100) { + + samples <- cells * genes + columns <- pull(slice_sample(as_tibble(object), n=cells), "cell") + rows <- pull(slice_sample(as_tibble(rowData(object)), n=genes), "name") + nonzero <- nnzero(counts(object)[rows, columns]) + sparsity <- (samples - nonzero) / samples + return(sparsity) + +} + +# the `replicate` function allows us to apply this many times over: +estimates <- replicate(n=100, sample_sparsity(tidybarnyard)) # "do it 100 times" + +# plot estimates: +library(ggplot2) +ggplot(tibble(estimate=estimates), aes(estimate)) + + geom_histogram() + + geom_vline(xintercept=sparsity, color="red", lwd=3) + + theme_minimal() + + ggtitle("Sparsity of UMI matrix (true value in red)") + +# the Central Limit Theorem lives to fight another day, +# and we have a decent idea of how to navigate our data. +``` + +Often, when someone cracks open a single cell dataset, the first thing they do +is to cluster the cells (based on whatever approach is fashionable). We don't +need to do that here. In fact, if you do plot the usual UMAP'ed clusters, +you'll mostly just see a batch effect. This takes a while to compute, so I +recommend you not bother with the next chunk. + +```{r, UMAP, eval=FALSE} + +library(scater) +# it is standard to log-normalize counts +# (although it's not actually a great idea) +tidybarnyard <- logNormCounts(tidybarnyard) + +# compute UMAP embedding on the most variable genes +tidybarnyard %>% runUMAP(ncomponents=3) -> tidyUMAP + +# plot using plotly, color by method +tidyUMAP %>% + plot_ly( + x =~ `UMAP1`, + y =~ `UMAP2`, + z =~ `UMAP3`, + color =~ method + ) ``` -Let's set up some thresholds for classifying cells as mouse or human. First, we -need to determine what fraction of transcripts from each genome each cell is -expressing. This is a job for a little function. +For this project, we know that the reads from each library (cell) were +competitively aligned against the mouse and human genomes. So all we really +need to do is to decide what we'll call a "mouse cell", what we'll call a +"human cell", and which we aren't confident calling either (unclassifiable). + +Originally, I wrote a little function to calculate what fraction of transcripts +from a genome were present in a given cell, and then made a matrix out of the +results. But since we already have a place to store the per-cell results, +why not just use that instead? + ```{r, bygenome} -genomes <- c(human="hg19", mouse="mm10") -fracExpressed <- function(g, object) { - subs <- subset(object, rowData(object)$genome == g) - colSums(counts(subs) > 0) / nrow(subs) -} +# identify the mouse genes: +as_tibble(rowData(tidybarnyard)) %>% # make a tibble from the rowData + filter(genome == "mm10") %>% # select just the mouse mm10 genes + pull("name") -> mouseGenes # assign their name column to mouseGenes + +# for technical reasons, it's faster to tally expressed genes this way: +tidybarnyard$fracmouse <- + (colSums(counts(tidybarnyard)[mouseGenes, ] > 0)) / length(mouseGenes) + +# do the same thing but with human genes: +as_tibble(rowData(tidybarnyard)) %>% # make a tibble from the rowData + filter(genome == "hg19") %>% # select just the human hg19 genes + pull("name") -> humanGenes # assign their name column to humanGenes + +# same remarks as previously +tidybarnyard$frachuman <- + (colSums(counts(tidybarnyard)[humanGenes, ] > 0)) / length(humanGenes) + +# why normalize by gene count? +as_tibble(rowData(tidybarnyard)) %>% select("genome") %>% table + + +# let's use ggplot to make sense of the results: +barnyardtibble <- as_tibble(tidybarnyard) + +p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.75, color="lightblue") + + geom_density2d(alpha=0.5, color="blue") + + theme_minimal() + +# first pass at a plot: +p + ggtitle("Barnyard experiment") -fracByCell <- do.call(cbind, - lapply(genomes, fracExpressed, object=tidybarnyard)) -smoothScatter(fracByCell) ``` -Suppose we want to implement some logic for gating cells. For example, we might say "if a cell has greater than 0.03 human transcriptomes expressed, less than 0.2 human transcriptomes, and less than 0.2 mouse transcriptomes, we'll call it human". Needless to say this is a bit of a craft. Let's implement it. - +# Gating + +Suppose we want to implement some logic for "gating" cells. For example, +we might say "if a cell has greater than 0.05 human transcriptomes expressed, +but less than 0.05 mouse transcriptomes, we'll call it human; if vice versa, +we'll call it a mouse cell; and if it has more than 0.05 of each, it's suspect". + +Needless to say this can stand a bit of exploration. Originally, I thought I'd +need to resort to writing functions for this purpose. Now I think it's actually +better to do it in a 'tidy' fashion. Let's use `mutate` for this purpose. + ```{r, gating} -# could do this "tidy" style on a tibble -is_human <- function(x, minHuman=0.03, maxHuman=0.2, maxMouse=0.03) { - if (x['mouse'] > maxMouse) return(FALSE) - else if (x['human'] > minHuman & x['human'] < maxHuman) return(TRUE) - else return(FALSE) -} - -# note redudancy -is_mouse <- function(x, minMouse=0.03, maxMouse=0.2, maxHuman=0.03) { - if (x['human'] > maxHuman) return(FALSE) -  else if (x['mouse'] > minMouse & x['mouse'] < maxMouse) return(TRUE) -  else return(FALSE) -} - -classified <- function(x) { - if (is_mouse(x) & is_human(x)) return(FALSE) - else if (is_mouse(x) | is_human(x)) return(TRUE) - else return(FALSE) -} - -frac <- data.frame(fracByCell) -frac$classifiable <- apply(frac, 1, classified) +# arbitrarily: +minhuman <- 0.05 +minmouse <- 0.05 +# Let's label any cell that is more than 5% (0.05) of BOTH genomes as suspect. + +# add a label: +barnyardtibble %>% + mutate(label = + case_when(frachuman > minhuman & fracmouse < minmouse ~ "human", + fracmouse > minmouse & frachuman < minhuman ~ "mouse", + TRUE ~ "suspect")) -> barnyardtibble + +# add our initial stab at labeling: +p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.75) + + theme_minimal() + +# plot it with some gates and density lines: +p + geom_density2d(alpha=0.5, color="blue") + + geom_vline(xintercept=minmouse, color="red") + + geom_hline(yintercept=minhuman, color="red") + + ggtitle("First stab at gating") + +``` + +This is looking a little better, but the density plots suggest there's a "hump" +at about 19% mouse or about 21% human above which the cells look like doublets. + +```{r, doubletgate} + +# arbitrarily: +maxhuman <- 0.21 +maxmouse <- 0.19 + +# make it obvious which cells are going to be gated out if we do this: +barnyardtibble %>% + mutate(doublet = frachuman > maxhuman | fracmouse > maxmouse) %>% + mutate(shading = case_when(label == "suspect" ~ 0.3, + doublet == TRUE ~ 0.1, + TRUE ~ 0.5)) -> barnyardtibble + +# start like the previous plot, but add low and high "gates" for each species: +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=label, alpha=I(shading))) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point() + + geom_segment(y=0, yend=minhuman, x=minmouse, xend=minmouse, color="black") + + geom_segment(y=0, yend=minhuman, x=maxmouse, xend=maxmouse, color="black") + + geom_segment(y=minhuman, yend=minhuman, x=minmouse, xend=maxmouse, + color="black") + + geom_segment(y=minhuman, yend=minhuman, x=0, xend=minmouse, color="black") + + geom_segment(y=maxhuman, yend=maxhuman, x=0, xend=minmouse, color="black") + + geom_segment(y=minhuman, yend=maxhuman, x=minmouse, xend=minmouse, + color="black") + + theme_minimal() + +# plot it +p + ggtitle("aggregate doublet gating") + + +``` + +Before we gate those out, let's see if this is related to library prep method: + +```{r, byMethod} + +# reuse the plot again: +p + facet_wrap(~ method) + +``` + +On second thought, let's gate on the minimums instead of throwing away cells. +We know this isn't a perfect approach to choosing a library prep method, but +it's surprisingly less bad than many approaches ("what's fashionable?", etc). + +```{r, regressFactors} + +# we need a 0/1 outcome to perform logistic regression: +barnyardtibble %>% mutate(classifiable = label != "suspect") -> barnyardtibble + +# logistic regression in R uses the glm() or general linear model function, +# with a binomial (0/1) link, and the result can be compared like any lm(): +fit0 <- glm(classifiable ~ 1, data=barnyardtibble, family=binomial) # null model + +# add `method` to the predictors +fit1 <- update(fit0, classifiable ~ method) + +# add `method` and `experiment` to the predictors +fit2 <- update(fit1, classifiable ~ method + experiment) + +# add `method` and `experiment` to the predictors, no intercept +fit3 <- update(fit2, classifiable ~ method + experiment + 0) + +# add `method` as the sole predictor, no intercept +fit4 <- update(fit0, classifiable ~ method + 0) + +``` + +Remember that logistic regression transforms the input probabilities, and that +means the coefficient estimates transform too. (Specifically, via `logit()`.) +In order to estimate the impact of a predictor back in "normal" space, you need +to expit (i.e., inverse logit) the values. Happily, the `coefplot` package will +do this for you, and the results can be interpreted as "what are the odds that +I will be able to classify a given cell with this method". + +```{r, coefficients} + +# it's better to use confidence intervals than p-values for fitting purposes, +# and it's even better yet to plot them all: +library(coefplot) + +# classifiable ~ method +coefplot(fit1, trans=invlogit) + theme_minimal() + +# classifiable ~ method + experiment +coefplot(fit2, trans=invlogit) + theme_minimal() + +# classifiable ~ method + experiment, no intercept +coefplot(fit3, trans=invlogit) + theme_minimal() + +# classifiable ~ method, no intercept +coefplot(fit4, trans=invlogit) + theme_minimal() + +``` + +Any thoughts on which library prep you'd use if cost is no object? + +# Mixture models as an alternative to manual gating + +Incidentally, we could also use a mixture model to classify all the points, and +in fact that is how I'd usually do it. One of my lab members has been working on +automatic gating of actual flow cytometry data this way, in fact, and it works +*great*. So without further ado... + +```{r, mixtureModel} + +# one of the greatest software packages ever written, +# fits a Gaussian mixture model with arbitrary covariance structure and uses +# a Bayesian penalization scheme to choose how many components exist in the mix +library(mclust) + +# since these are proportional values, it makes sense to transform them: +mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), G=1:3) +# here I have restricted Mclust to fitting, at most, 3 components (G=1:3). +# this speeds up the process and avoids some difficult questions later on ;-) + +# create a new column of the barnyard tibble with the results: +table(mfit$classification) # it turns out that we end up with less human cells +barnyardtibble$mclass <- ifelse(mfit$classification == 2, "suspect", + ifelse(mfit$classification == 1, "human", + "mouse")) # not sure how do this tidily. + +# add mixture assignments: +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.5) + + theme_minimal() + +# plot it +p + ggtitle("mixture model fit") + +``` + +Suppose we re-run the regressions using the mixture model fits. What happens? + +```{r, remix} + +barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble + +# null model +fitm0 <- glm(mclassifiable ~ 0, data=barnyardtibble, family=binomial) # random + +# regress `mclassifiable` on method, no intercept: +fitm1 <- update(fitm0, mclassifiable ~ method + 0) +coefplot(fitm1, trans=invlogit) + theme_minimal() + +# regress `mclassifiable` on method and experiment, no intercept: +fitm2 <- update(fitm0, mclassifiable ~ method + experiment + 0) +coefplot(fitm2, trans=invlogit) + theme_minimal() + +# regress `mclassifiable` on method interacting with experiment, no intercept: +fitm3 <- update(fitm0, mclassifiable ~ method * experiment + 0) +coefplot(fitm3, trans=invlogit) + theme_minimal() - ``` - -_Question: Can you do this again but for each method?_ -_Question: Can you do this again but for each mixture?_ +Have your thoughts on which method to use changed? Remember, each coefficient +ends up being the odds that a cell can be classified, so you have to multiply +through the values that are true for each cell. Given the confidence intervals +for experiment and the interaction terms, do they add much to the model? + +You *could* do this fit via ANOVA, but I claim it's a bit easier to interpret +the odds (multiply by 100 to get percent chance of classification!) from LR. +One thing you might consider is to allow Mclust to use more possible values for +G (the number of Groups). Its default is "up to 10". If you plot the results, +do you still feel comfortable interpreting one of the groups as "suspect"? Is +it still reasonable to use _glm_ (for logistic regression) in that case? 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classification and plotting +# Background Recall that we are seeking to identify the influence of two factors on our ability to classify cells: the experimental vial and the library prep protocol. @@ -28,16 +28,7 @@ let us answer ANOVA-style questions: "how much variability is explained by a particular factor in the model?" First we need to set up our response variable, i.e., the ability to classify a cell confidently. For that, we need cutoffs. -![bus format](../figure/bustools.jpeg) - -The barcode, UMI, set or [BUS format](https://academic.oup.com/bioinformatics/article/35/21/4472/5487510) neatly encapsulates the process of transforming reads -resulting from a single-cell RNAseq experiment into counts of molecular barcodes -describing a cell, a molecule, and the likely genomic origin of that molecule. -It's one of the most compact representations possible for raw scRNAseq outputs, -and the figure shows how it facilitates direct comparisons. The Broad used their -`scumi` tool instead, but the basic notion ("make everything comparable") and -the resulting sparse matrix of counts is similar in spirit. - +# tidySingleCellExperiment With the release of Bioconductor 3.14, the project includes a tidy single cell experiment (data structure) package, which is great since all other single cell @@ -73,6 +64,7 @@ You could always adjust the `ideal` argument to the function below to resample: # adapted from a SingleCellExperiment-centric method for CITEseq sample_umis <- function(umis, meta, block, ideal=300) { + # {{{ stopifnot(nrow(meta) == ncol(umis)) stopifnot(length(block) == nrow(meta)) @@ -99,44 +91,625 @@ sample_umis <- function(umis, meta, block, ideal=300) { message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), " cells in ", length(samplesets), " blocks.") umis[, keep] + # }}} } ``` -Load the data: +# Tim's stab at classification and plotting + +It will be difficult to evaluate our strategy if we don't first load the data: ```r library(SingleCellExperiment) +#> Loading required package: SummarizedExperiment +#> Loading required package: MatrixGenerics +#> Loading required package: matrixStats +#> +#> Attaching package: 'matrixStats' +#> The following object is masked from 'package:dplyr': +#> +#> count +#> +#> Attaching package: 'MatrixGenerics' +#> The following objects are masked from 'package:matrixStats': +#> +#> colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse, +#> colCounts, colCummaxs, colCummins, colCumprods, colCumsums, +#> colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs, +#> colMads, colMaxs, colMeans2, colMedians, colMins, colOrderStats, +#> colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds, +#> colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads, +#> colWeightedMeans, colWeightedMedians, colWeightedSds, +#> colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet, +#> rowCollapse, rowCounts, rowCummaxs, rowCummins, rowCumprods, +#> rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps, +#> rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians, rowMins, +#> rowOrderStats, rowProds, rowQuantiles, rowRanges, rowRanks, +#> rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs, rowVars, +#> rowWeightedMads, rowWeightedMeans, rowWeightedMedians, +#> rowWeightedSds, rowWeightedVars +#> Loading required package: GenomicRanges +#> Loading required package: stats4 +#> Loading required package: BiocGenerics +#> +#> Attaching package: 'BiocGenerics' +#> The following objects are masked from 'package:stats': +#> +#> IQR, mad, sd, var, xtabs +#> The following objects are masked from 'package:dplyr': +#> +#> combine, intersect, setdiff, union +#> The following objects are masked from 'package:base': +#> +#> Filter, Find, Map, Position, Reduce, anyDuplicated, append, +#> as.data.frame, basename, cbind, colnames, dirname, do.call, +#> duplicated, eval, evalq, get, grep, grepl, intersect, is.unsorted, +#> lapply, mapply, match, mget, order, paste, pmax, pmax.int, pmin, +#> pmin.int, rank, rbind, rownames, sapply, setdiff, sort, table, +#> tapply, union, unique, unsplit, which.max, which.min +#> Loading required package: S4Vectors +#> +#> Attaching package: 'S4Vectors' +#> The following object is masked from 'package:tidyr': +#> +#> expand +#> The following objects are masked from 'package:dplyr': +#> +#> first, rename +#> The following objects are masked from 'package:base': +#> +#> I, expand.grid, unname +#> Loading required package: IRanges +#> +#> Attaching package: 'IRanges' +#> The following object is masked from 'package:purrr': +#> +#> reduce +#> The following objects are masked from 'package:dplyr': +#> +#> collapse, desc, slice +#> Loading required package: GenomeInfoDb +#> Loading required package: Biobase +#> Welcome to Bioconductor +#> +#> Vignettes contain introductory material; view with +#> 'browseVignettes()'. To cite Bioconductor, see +#> 'citation("Biobase")', and for packages 'citation("pkgname")'. +#> +#> Attaching package: 'Biobase' +#> The following object is masked from 'package:MatrixGenerics': +#> +#> rowMedians +#> The following objects are masked from 'package:matrixStats': +#> +#> anyMissing, rowMedians library(tidySingleCellExperiment) +# should package this... if (!exists("tidybarnyard")) tidybarnyard <- tidy(readRDS(url("https://ttriche.github.io/RDS/barnyard.rds"))) -rowGenome <- rowData(tidybarnyard)$genome +#> Warning: `tidy()` was deprecated in tidySingleCellExperiment 1.1.1. +#> tidySingleCellExperiment says: tidy() is not needed anymore. +#> This warning is displayed once every 8 hours. +#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated. ``` - -Let's set up some thresholds for classifying cells as mouse or human. First, we -need to determine what fraction of transcripts from each genome each cell is -expressing. This is a job for a little function. +It will be useful to know that, when you use the `$` operator on a typical +Bioconductor object (such as a _tidySingleCellExperiment_ like ours), it assumes +you want to see the column data (`colData`) with that name (see below). For +example, when we built up the `tidybarnyard` object from the cell and gene +tables (by splitting and mutating them), we fed them to the SingleCellExperiment +constructor function and it bolted them onto the side of the object. It turns +out _[SingleCellExperiment](https://www.nature.com/articles/s41592-019-0654-x)_ +is just a modified version of _[SummarizedExperiment](https://www.nature.com/articles/nmeth.3252)_: + +![the SummarizedExperiment scheme](../figure/SummarizedExperiment.png) + +The figure from the original paper is better/easier to understand, although you +can certainly read about the new one. TidySingleCellExperiment wraps the "newer" +_[SingleCellExperiment](https://www.nature.com/articles/s41592-019-0654-x)_ +so that it plays somewhat more nicely with the tidyverse we've all been using. +There are many other approaches to analyzing single-cell data, and you may well +bump into several of them, but this one happens to be very efficient. Plus, +keeping sample- or cell-specific details lined up with columns, and gene- or +feature-specific details lined up with rows, tends to stay in style. The only +annoying feature (ha!) with this setup is that you need to explicitly tell R +when you want data about the rows themselves (i.e., the rowData). + +The _[tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html)_ package gives +us some tools to stay in the Tidyverse as much as possible while working with +single-cell data. (It turns out that the underlying data makes this somewhat +less trivial than you might expect. Fortunately, you don't have to care.) + +Concretely, let's see how these moving parts fit together. The `dim` function +provides dimensions for a rectangular object, rows x columns, which makes it +a little easier to keep track of what's going on when we subset either one. ```r -genomes <- c(human="hg19", mouse="mm10") -fracExpressed <- function(g, object) { - subs <- subset(object, rowData(object)$genome == g) - colSums(counts(subs) > 0) / nrow(subs) +# as what is our object masquerading? +show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" +#> # A SingleCellExperiment-tibble abstraction: 0 × 5 +#> # Features=62046 | Assays=counts +#> # … with 5 variables: cell , name , experiment , method , +#> # cell + +# how many rows (genes) and columns (cells) are there in our tidy barnyard? +dim(tidybarnyard) +#> [1] 62046 4199 + +# is this equivalent to what we get from nrow() and ncol()? +identical(dim(tidybarnyard), + c(nrow(tidybarnyard), ncol(tidybarnyard))) +#> [1] TRUE + +# the assay(s) should also have this many rows and columns. Do they? +identical(dim(tidybarnyard), dim(assay(tidybarnyard))) +#> [1] TRUE + +# there is a shortcut for the `counts` assay, since it's so common: +counts(tidybarnyard)[1:3, 1:3] +#> 3 x 3 sparse Matrix of class "dgCMatrix" +#> Mixture1.10x-Chromium-v2.GGGCATCGTCACACGC +#> hg19_ENSG00000000003_hg19_TSPAN6 1 +#> hg19_ENSG00000000005_hg19_TNMD . +#> hg19_ENSG00000000419_hg19_DPM1 2 +#> Mixture1.10x-Chromium-v2.CACATAGAGATACACA +#> hg19_ENSG00000000003_hg19_TSPAN6 . +#> hg19_ENSG00000000005_hg19_TNMD . +#> hg19_ENSG00000000419_hg19_DPM1 . +#> Mixture1.10x-Chromium-v2.CACTCCATCCTCCTAG +#> hg19_ENSG00000000003_hg19_TSPAN6 . +#> hg19_ENSG00000000005_hg19_TNMD . +#> hg19_ENSG00000000419_hg19_DPM1 . + +# does each row (gene) in the object have a corresponding rowData() row? +identical(rownames(tidybarnyard), rownames(rowData(tidybarnyard))) +#> [1] TRUE + +# does each column (cell) in the object have a corresponding colData() row? +identical(colnames(tidybarnyard), rownames(colData(tidybarnyard))) +#> [1] TRUE + +# is there a rowData row and a colData row that corresponds to each UMI count? +identical(dim(counts(tidybarnyard)), + c(nrow(rowData(tidybarnyard)), nrow(colData(tidybarnyard)))) +#> [1] TRUE + +# does that mean we can ask for a random cell with certain attributes? +as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" + filter(method == "inDrops") %>% # "choose rows where method == inDrops" + slice_sample %>% # "randomly slice out one row" + pull("cell") -> aCell # "pull the column cell, assign to aCell" +#> Error: Can't transform a data frame with duplicate names. + +# your cell: +show(aCell) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'show': object 'aCell' not found + +# does that mean we can ask for a random gene with certain attributes? +as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" + filter(genome == "mm10") %>% # "choose rows where genome == mm10" + slice_sample %>% # "randomly slice out one row" + pull("name") -> aGene # "pull the column name, assign to aGene" + +# your gene: +show(aGene) +#> [1] "mm10_ENSMUSG00000098847_mm10_Gm27235" + +# how many copies of this random gene were found in this random cell? +counts(tidybarnyard)[aGene, aCell] +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'j' in selecting a method for function '[': object 'aCell' not found + +# note that the odds are good that you'll get a 0 for this random combination: +library(Matrix) # for the `nnzero` function +sparsity <- 1 - (nnzero(counts(tidybarnyard)) / # number of nonzero counts + prod(dim(tidybarnyard))) # number of rows * columns + +# specifically, the chance of getting a zero is about 92.3%: +message(round(sparsity * 100, 1), "%") +#> 92.3% + +# This is fairly typical for a single-cell experiment, perhaps a bit low even. +# You could also take 10000 or so random samples to estimate the sparsity. +# For example, you could grab 100 genes from 100 cells at a time: +as_tibble(tidybarnyard) %>% # turn tidybarnyard's colData into a tibble + slice_sample(n=100) %>% # slice out 100 random rows, and then... + pull("cell") -> aHundredCells # assign the "cell" column to "aHundredCells" +#> Error: Can't transform a data frame with duplicate names. + +as_tibble(rowData(tidybarnyard)) %>% # turn tidybarnyard's rowData into a tibble + slice_sample(n=100) %>% # slice out 100 random rows, and then + pull("name") -> aHundredGenes # assign "name" column to "aHundredGenes" + +samples <- (length(aHundredCells) * length(aHundredGenes)) +#> Error in eval(expr, envir, enclos): object 'aHundredCells' not found +nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'x' in selecting a method for function 'nnzero': error in evaluating the argument 'j' in selecting a method for function '[': object 'aHundredCells' not found +sparsity_hat <- (samples - nonzero) / samples +#> Error in eval(expr, envir, enclos): object 'nonzero' not found +sparsity_hat # estimated sparsity +#> Error in eval(expr, envir, enclos): object 'sparsity_hat' not found + +# In fact, we can use this scheme to look at sampling error: +sample_sparsity <- function(object, cells=100, genes=100) { + + samples <- cells * genes + columns <- pull(slice_sample(as_tibble(object), n=cells), "cell") + rows <- pull(slice_sample(as_tibble(rowData(object)), n=genes), "name") + nonzero <- nnzero(counts(object)[rows, columns]) + sparsity <- (samples - nonzero) / samples + return(sparsity) + } -fracByCell <- do.call(cbind, - lapply(genomes, fracExpressed, object=tidybarnyard)) -smoothScatter(fracByCell) +# the `replicate` function allows us to apply this many times over: +estimates <- replicate(n=100, sample_sparsity(tidybarnyard)) # "do it 100 times" +#> Error: Can't transform a data frame with duplicate names. + +# plot estimates: +library(ggplot2) +ggplot(tibble(estimate=estimates), aes(estimate)) + + geom_histogram() + + geom_vline(xintercept=sparsity, color="red", lwd=3) + + theme_minimal() + + ggtitle("Sparsity of UMI matrix (true value in red)") +#> Error in eval_tidy(xs[[j]], mask): object 'estimates' not found + +# the Central Limit Theorem lives to fight another day, +# and we have a decent idea of how to navigate our data. +``` + +Often, when someone cracks open a single cell dataset, the first thing they do +is to cluster the cells (based on whatever approach is fashionable). We don't +need to do that here. In fact, if you do plot the usual UMAP'ed clusters, +you'll mostly just see a batch effect. This takes a while to compute, so I +recommend you not bother with the next chunk. + + +```r + +library(scater) +# it is standard to log-normalize counts +# (although it's not actually a great idea) +tidybarnyard <- logNormCounts(tidybarnyard) + +# compute UMAP embedding on the most variable genes +tidybarnyard %>% runUMAP(ncomponents=3) -> tidyUMAP + +# plot using plotly, color by method +tidyUMAP %>% + plot_ly( + x =~ `UMAP1`, + y =~ `UMAP2`, + z =~ `UMAP3`, + color =~ method + ) + +``` + +For this project, we know that the reads from each library (cell) were +competitively aligned against the mouse and human genomes. So all we really +need to do is to decide what we'll call a "mouse cell", what we'll call a +"human cell", and which we aren't confident calling either (unclassifiable). + +Originally, I wrote a little function to calculate what fraction of transcripts +from a genome were present in a given cell, and then made a matrix out of the +results. But since we already have a place to store the per-cell results, +why not just use that instead? + + + +```r + +# identify the mouse genes: +as_tibble(rowData(tidybarnyard)) %>% # make a tibble from the rowData + filter(genome == "mm10") %>% # select just the mouse mm10 genes + pull("name") -> mouseGenes # assign their name column to mouseGenes + +# for technical reasons, it's faster to tally expressed genes this way: +tidybarnyard$fracmouse <- + (colSums(counts(tidybarnyard)[mouseGenes, ] > 0)) / length(mouseGenes) + +# do the same thing but with human genes: +as_tibble(rowData(tidybarnyard)) %>% # make a tibble from the rowData + filter(genome == "hg19") %>% # select just the human hg19 genes + pull("name") -> humanGenes # assign their name column to humanGenes + +# same remarks as previously +tidybarnyard$frachuman <- + (colSums(counts(tidybarnyard)[humanGenes, ] > 0)) / length(humanGenes) + +# why normalize by gene count? +as_tibble(rowData(tidybarnyard)) %>% select("genome") %>% table +#> . +#> hg19 mm10 +#> 33354 28692 + + +# let's use ggplot to make sense of the results: +barnyardtibble <- as_tibble(tidybarnyard) + +p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.75, color="lightblue") + + geom_density2d(alpha=0.5, color="blue") + + theme_minimal() + +# first pass at a plot: +p + ggtitle("Barnyard experiment") +#> Error: `data` must be uniquely named but has duplicate columns ``` ![plot of chunk bygenome](figure/bygenome-1.png) -_Question: Can you do this again but for each method?_ +# Gating + +Suppose we want to implement some logic for "gating" cells. For example, +we might say "if a cell has greater than 0.05 human transcriptomes expressed, +but less than 0.05 mouse transcriptomes, we'll call it human; if vice versa, +we'll call it a mouse cell; and if it has more than 0.05 of each, it's suspect". + +Needless to say this can stand a bit of exploration. Originally, I thought I'd +need to resort to writing functions for this purpose. Now I think it's actually +better to do it in a 'tidy' fashion. Let's use `mutate` for this purpose. + + +```r + +# arbitrarily: +minhuman <- 0.05 +minmouse <- 0.05 +# Let's label any cell that is more than 5% (0.05) of BOTH genomes as suspect. + +# add a label: +barnyardtibble %>% + mutate(label = + case_when(frachuman > minhuman & fracmouse < minmouse ~ "human", + fracmouse > minmouse & frachuman < minhuman ~ "mouse", + TRUE ~ "suspect")) -> barnyardtibble +#> Error: Can't transform a data frame with duplicate names. + +# add our initial stab at labeling: +p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.75) + + theme_minimal() + +# plot it with some gates and density lines: +p + geom_density2d(alpha=0.5, color="blue") + + geom_vline(xintercept=minmouse, color="red") + + geom_hline(yintercept=minhuman, color="red") + + ggtitle("First stab at gating") +#> Error: `data` must be uniquely named but has duplicate columns +``` + +![plot of chunk gating](figure/gating-1.png) + +This is looking a little better, but the density plots suggest there's a "hump" +at about 19% mouse or about 21% human above which the cells look like doublets. + + +```r + +# arbitrarily: +maxhuman <- 0.21 +maxmouse <- 0.19 + +# make it obvious which cells are going to be gated out if we do this: +barnyardtibble %>% + mutate(doublet = frachuman > maxhuman | fracmouse > maxmouse) %>% + mutate(shading = case_when(label == "suspect" ~ 0.3, + doublet == TRUE ~ 0.1, + TRUE ~ 0.5)) -> barnyardtibble +#> Error: Can't transform a data frame with duplicate names. + +# start like the previous plot, but add low and high "gates" for each species: +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=label, alpha=I(shading))) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point() + + geom_segment(y=0, yend=minhuman, x=minmouse, xend=minmouse, color="black") + + geom_segment(y=0, yend=minhuman, x=maxmouse, xend=maxmouse, color="black") + + geom_segment(y=minhuman, yend=minhuman, x=minmouse, xend=maxmouse, + color="black") + + geom_segment(y=minhuman, yend=minhuman, x=0, xend=minmouse, color="black") + + geom_segment(y=maxhuman, yend=maxhuman, x=0, xend=minmouse, color="black") + + geom_segment(y=minhuman, yend=maxhuman, x=minmouse, xend=minmouse, + color="black") + + theme_minimal() + +# plot it +p + ggtitle("aggregate doublet gating") +#> Error: `data` must be uniquely named but has duplicate columns +``` + +![plot of chunk doubletgate](figure/doubletgate-1.png) + +Before we gate those out, let's see if this is related to library prep method: + + +```r + +# reuse the plot again: +p + facet_wrap(~ method) +#> Error: `data` must be uniquely named but has duplicate columns +``` + +![plot of chunk byMethod](figure/byMethod-1.png) + +On second thought, let's gate on the minimums instead of throwing away cells. +We know this isn't a perfect approach to choosing a library prep method, but +it's surprisingly less bad than many approaches ("what's fashionable?", etc). + + +```r + +# we need a 0/1 outcome to perform logistic regression: +barnyardtibble %>% mutate(classifiable = label != "suspect") -> barnyardtibble +#> Error: Can't transform a data frame with duplicate names. + +# logistic regression in R uses the glm() or general linear model function, +# with a binomial (0/1) link, and the result can be compared like any lm(): +fit0 <- glm(classifiable ~ 1, data=barnyardtibble, family=binomial) # null model +#> Error in eval(predvars, data, env): object 'classifiable' not found + +# add `method` to the predictors +fit1 <- update(fit0, classifiable ~ method) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit0' not found + +# add `method` and `experiment` to the predictors +fit2 <- update(fit1, classifiable ~ method + experiment) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit1' not found + +# add `method` and `experiment` to the predictors, no intercept +fit3 <- update(fit2, classifiable ~ method + experiment + 0) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit2' not found + +# add `method` as the sole predictor, no intercept +fit4 <- update(fit0, classifiable ~ method + 0) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit0' not found +``` + +Remember that logistic regression transforms the input probabilities, and that +means the coefficient estimates transform too. (Specifically, via `logit()`.) +In order to estimate the impact of a predictor back in "normal" space, you need +to expit (i.e., inverse logit) the values. Happily, the `coefplot` package will +do this for you, and the results can be interpreted as "what are the odds that +I will be able to classify a given cell with this method". + + +```r + +# it's better to use confidence intervals than p-values for fitting purposes, +# and it's even better yet to plot them all: +library(coefplot) + +# classifiable ~ method +coefplot(fit1, trans=invlogit) + theme_minimal() +#> Error in coefplot(fit1, trans = invlogit): object 'fit1' not found + +# classifiable ~ method + experiment +coefplot(fit2, trans=invlogit) + theme_minimal() +#> Error in coefplot(fit2, trans = invlogit): object 'fit2' not found + +# classifiable ~ method + experiment, no intercept +coefplot(fit3, trans=invlogit) + theme_minimal() +#> Error in coefplot(fit3, trans = invlogit): object 'fit3' not found + +# classifiable ~ method, no intercept +coefplot(fit4, trans=invlogit) + theme_minimal() +#> Error in coefplot(fit4, trans = invlogit): object 'fit4' not found +``` + +Any thoughts on which library prep you'd use if cost is no object? + +# Mixture models as an alternative to manual gating + +Incidentally, we could also use a mixture model to classify all the points, and +in fact that is how I'd usually do it. One of my lab members has been working on +automatic gating of actual flow cytometry data this way, in fact, and it works +*great*. So without further ado... + + +```r + +# one of the greatest software packages ever written, +# fits a Gaussian mixture model with arbitrary covariance structure and uses +# a Bayesian penalization scheme to choose how many components exist in the mix +library(mclust) +#> __ ___________ __ _____________ +#> / |/ / ____/ / / / / / ___/_ __/ +#> / /|_/ / / / / / / / /\__ \ / / +#> / / / / /___/ /___/ /_/ /___/ // / +#> /_/ /_/\____/_____/\____//____//_/ version 5.4.8 +#> Type 'citation("mclust")' for citing this R package in publications. + +# since these are proportional values, it makes sense to transform them: +mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), G=1:3) +#> fitting ... +#> | | | 0% | |== | 2% | |=== | 5% | |===== | 7% | |======= | 9% | |======== | 12% | |========== | 14% | |=========== | 16% | |============= | 19% | |=============== | 21% | |================ | 23% | |================== | 26% | |==================== | 28% | |===================== | 30% | |======================= | 33% | |======================== | 35% | |========================== | 37% | |============================ | 40% | |============================= | 42% | |=============================== | 44% | |================================= | 47% | |================================== | 49% | |==================================== | 51% | |===================================== | 53% | |======================================= | 56% | |========================================= | 58% | |========================================== | 60% | |============================================ | 63% | |============================================== | 65% | |=============================================== | 67% | |================================================= | 70% | |================================================== | 72% | |==================================================== | 74% | |====================================================== | 77% | |======================================================= | 79% | |========================================================= | 81% | |=========================================================== | 84% | |============================================================ | 86% | |============================================================== | 88% | |=============================================================== | 91% | |================================================================= | 93% | |=================================================================== | 95% | |==================================================================== | 98% | |======================================================================| 100% +# here I have restricted Mclust to fitting, at most, 3 components (G=1:3). +# this speeds up the process and avoids some difficult questions later on ;-) + +# create a new column of the barnyard tibble with the results: +table(mfit$classification) # it turns out that we end up with less human cells +#> +#> 1 2 3 +#> 2116 1941 142 +barnyardtibble$mclass <- ifelse(mfit$classification == 2, "suspect", + ifelse(mfit$classification == 1, "human", + "mouse")) # not sure how do this tidily. + +# add mixture assignments: +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.5) + + theme_minimal() + +# plot it +p + ggtitle("mixture model fit") +#> Error: `data` must be uniquely named but has duplicate columns +``` + +![plot of chunk mixtureModel](figure/mixtureModel-1.png) + +Suppose we re-run the regressions using the mixture model fits. What happens? + + +```r + +barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble +#> Error: Can't transform a data frame with duplicate names. + +# null model +fitm0 <- glm(mclassifiable ~ 0, data=barnyardtibble, family=binomial) # random +#> Error in eval(predvars, data, env): object 'mclassifiable' not found + +# regress `mclassifiable` on method, no intercept: +fitm1 <- update(fitm0, mclassifiable ~ method + 0) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fitm0' not found +coefplot(fitm1, trans=invlogit) + theme_minimal() +#> Error in coefplot(fitm1, trans = invlogit): object 'fitm1' not found + +# regress `mclassifiable` on method and experiment, no intercept: +fitm2 <- update(fitm0, mclassifiable ~ method + experiment + 0) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fitm0' not found +coefplot(fitm2, trans=invlogit) + theme_minimal() +#> Error in coefplot(fitm2, trans = invlogit): object 'fitm2' not found + +# regress `mclassifiable` on method interacting with experiment, no intercept: +fitm3 <- update(fitm0, mclassifiable ~ method * experiment + 0) +#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fitm0' not found +coefplot(fitm3, trans=invlogit) + theme_minimal() +#> Error in coefplot(fitm3, trans = invlogit): object 'fitm3' not found +``` -_Question: Can you do this again but for each mixture?_ +Have your thoughts on which method to use changed? Remember, each coefficient +ends up being the odds that a cell can be classified, so you have to multiply +through the values that are true for each cell. Given the confidence intervals +for experiment and the interaction terms, do they add much to the model? + +You *could* do this fit via ANOVA, but I claim it's a bit easier to interpret +the odds (multiply by 100 to get percent chance of classification!) from LR. +One thing you might consider is to allow Mclust to use more possible values for +G (the number of Groups). Its default is "up to 10". If you plot the results, +do you still feel comfortable interpreting one of the groups as "suspect"? Is +it still reasonable to use _glm_ (for logistic regression) in that case? From 59abc3a360bbefd65c69b8a916f95c599dbc2f2e Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 09:43:11 -0500 Subject: [PATCH 07/25] add updated run output --- .../project2_chunks/figure/byMethod-1.png | Bin 362 -> 52264 bytes .../project2_chunks/figure/bygenome-1.png | Bin 362 -> 79008 bytes .../project2_chunks/figure/coefficients-1.png | Bin 0 -> 16336 bytes .../project2_chunks/figure/coefficients-2.png | Bin 0 -> 17947 bytes .../project2_chunks/figure/coefficients-3.png | Bin 0 -> 18024 bytes .../project2_chunks/figure/coefficients-4.png | Bin 0 -> 16536 bytes .../project2_chunks/figure/doubletgate-1.png | Bin 362 -> 62468 bytes vignettes/project2_chunks/figure/gating-1.png | Bin 362 -> 77921 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 362 -> 32427 bytes vignettes/project2_chunks/figure/remix-1.png | Bin 0 -> 16317 bytes vignettes/project2_chunks/figure/remix-2.png | Bin 0 -> 18181 bytes vignettes/project2_chunks/figure/remix-3.png | Bin 0 -> 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zC||0VM{|R|y)kFLohBFfWUlo|Ti+*2eS(Cb4$niTb#=#f1=KJTWA z`FjY#V}eY|M@skt}ImucESqK6_T^g##|0mMw<>S(zYR_W3qV%#y&gX3xjeEo+VlZvMJS=lSGUf$PeTK|gHyES zcE=xeSW}(`KDlQx`i4T+)w>)L-563-t90OypSx4)j1S&#4_8?r#Etj$-lB3P!D4dj?)#bx9tf~{ za}yqBgV&ar0WSxAyrndk;@aZ3#fZ()%F|J1aMDULS!n#4 zGW(^F-?WI>r(C>!PXU&o4W3aeFSrVR%=|ETxc0m7?v*4Kx7r*0>yaReJ4{_2Rgk4F zlyuIU$b*kA;J4uHS9LIMw|TL7UDujHP=48(V6gS+Q|x4vehqHcF`>`c1ZEP!taQDl z<*9M&D*qpwAm`P;e+by+*D3%S*RjD;_OXZ4-j>yr3FyQA#G(n?Mvu(!O4q_E99Tqo z%wJHm0d+{e-wEP_j%-7@gI(Nm^B_Po-vwS6ZQG}x|6Z;m(Sdl-m4hsKW@E7&7^#{q zKGUxcH*9Ux5x)4mtG-s-QKa_tE#|$Oe7MWH|34R?1CO>uCb%Ryv6SZ*lHsu->wSXU zaT=+sFn>vmdc**C@WR(oY7$#S!=e#)-^Z*e;YJeb7F)*zin0 zHAeF=)JHDoQQyzjni7|>`@HFQnN@ldClick for details -```{r, tidysinglecell, eval=FALSE} +```{r, tidysinglecell} if (!require("SingleCellExperiment")) { BiocManager::install("SingleCellExperiment") @@ -56,64 +56,25 @@ if (!require("tidySingleCellExperiment")) { ``` -# Sort-of-bonus: resampling - -Above, I stated that maybe we don't need thousands of cells per method. -You could always adjust the `ideal` argument to the function below to resample: -
- Click for sample_umis() function code -```{r, sample_umis} - -# adapted from a SingleCellExperiment-centric method for CITEseq -sample_umis <- function(umis, meta, block, ideal=300) { - - # {{{ - stopifnot(nrow(meta) == ncol(umis)) - stopifnot(length(block) == nrow(meta)) - - pops <- sort(table(block)) - samplesets <- split(seq_len(nrow(meta)), block) - - keep <- integer() - for (set in names(samplesets)) { - sset <- samplesets[[set]] - cells <- length(sset) - if (cells <= ideal) { - pct <- 100 - keep <- c(keep, sset) - message("Kept ", cells, " cells (", pct, "%) of type ", set, ".") - } else { - kept <- sample(sset, size=ideal) - pct <- round((ideal / cells) * 100) - keep <- c(keep, kept) - message("Kept ", ideal, " cells (", pct, "%) of type ", set, ".") - } - } - - pct <- round((length(keep) / ncol(umis)) * 100, 1) - message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), - " cells in ", length(samplesets), " blocks.") - umis[, keep] - # }}} - -} -``` -
- # Tim's stab at classification and plotting It will be difficult to evaluate our strategy if we don't first load the data: -```{r, gettidysce } - +
+ Click for details +```{r, loadpackages} library(SingleCellExperiment) library(tidySingleCellExperiment) +``` +
-# should package this... -if (!exists("tidybarnyard")) -tidybarnyard <- tidy(readRDS(url("https://ttriche.github.io/RDS/barnyard.rds"))) - +```{r, gettidysce } +# should package this... could just instantiate from a package via data(...) +if (!exists("tidybarnyard")) { + tidybarnyard <- readRDS(url("https://ttriche.github.io/RDS/tidybarnyard.rds")) +} ``` + It will be useful to know that, when you use the `$` operator on a typical Bioconductor object (such as a _tidySingleCellExperiment_ like ours), it assumes you want to see the column data (`colData`) with that name (see below). For @@ -123,7 +84,7 @@ constructor function and it bolted them onto the side of the object. It turns out _[SingleCellExperiment](https://www.nature.com/articles/s41592-019-0654-x)_ is just a modified version of _[SummarizedExperiment](https://www.nature.com/articles/nmeth.3252)_: -![the SummarizedExperiment scheme](../figure/SummarizedExperiment.png) +![the SummarizedExperiment scheme](../figure/SummarizedExperiment.jpg) The figure from the original paper is better/easier to understand, although you can certainly read about the new one. TidySingleCellExperiment wraps the "newer" @@ -486,7 +447,8 @@ automatic gating of actual flow cytometry data this way, in fact, and it works library(mclust) # since these are proportional values, it makes sense to transform them: -mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), G=1:3) +mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), + verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! # here I have restricted Mclust to fitting, at most, 3 components (G=1:3). # this speeds up the process and avoids some difficult questions later on ;-) @@ -545,3 +507,48 @@ One thing you might consider is to allow Mclust to use more possible values for G (the number of Groups). Its default is "up to 10". If you plot the results, do you still feel comfortable interpreting one of the groups as "suspect"? Is it still reasonable to use _glm_ (for logistic regression) in that case? + +# Sort-of-bonus: resampling + +Above, I stated that maybe we don't need thousands of cells per method. +You could always adjust the `ideal` argument to the function below to resample: +
+ Click for sample_umis() function code +```{r, sample_umis} + +# adapted from a SingleCellExperiment-centric method for CITEseq +sample_umis <- function(umis, meta, block, ideal=300) { + + # {{{ + stopifnot(nrow(meta) == ncol(umis)) + stopifnot(length(block) == nrow(meta)) + + pops <- sort(table(block)) + samplesets <- split(seq_len(nrow(meta)), block) + + keep <- integer() + for (set in names(samplesets)) { + sset <- samplesets[[set]] + cells <- length(sset) + if (cells <= ideal) { + pct <- 100 + keep <- c(keep, sset) + message("Kept ", cells, " cells (", pct, "%) of type ", set, ".") + } else { + kept <- sample(sset, size=ideal) + pct <- round((ideal / cells) * 100) + keep <- c(keep, kept) + message("Kept ", ideal, " cells (", pct, "%) of type ", set, ".") + } + } + + pct <- round((length(keep) / ncol(umis)) * 100, 1) + message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), + " cells in ", length(samplesets), " blocks.") + umis[, keep] + # }}} + +} +``` +
+ diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 585a722..36742ec 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -48,155 +48,30 @@ if (!require("tidySingleCellExperiment")) { BiocManager::install("tidySingleCellExperiment") library(tidySingleCellExperiment) } - ``` -# Sort-of-bonus: resampling - -Above, I stated that maybe we don't need thousands of cells per method. -You could always adjust the `ideal` argument to the function below to resample: -
- Click for sample_umis() function code - -```r - -# adapted from a SingleCellExperiment-centric method for CITEseq -sample_umis <- function(umis, meta, block, ideal=300) { - - # {{{ - stopifnot(nrow(meta) == ncol(umis)) - stopifnot(length(block) == nrow(meta)) - - pops <- sort(table(block)) - samplesets <- split(seq_len(nrow(meta)), block) - - keep <- integer() - for (set in names(samplesets)) { - sset <- samplesets[[set]] - cells <- length(sset) - if (cells <= ideal) { - pct <- 100 - keep <- c(keep, sset) - message("Kept ", cells, " cells (", pct, "%) of type ", set, ".") - } else { - kept <- sample(sset, size=ideal) - pct <- round((ideal / cells) * 100) - keep <- c(keep, kept) - message("Kept ", ideal, " cells (", pct, "%) of type ", set, ".") - } - } - - pct <- round((length(keep) / ncol(umis)) * 100, 1) - message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), - " cells in ", length(samplesets), " blocks.") - umis[, keep] - # }}} - -} -``` -
- # Tim's stab at classification and plotting It will be difficult to evaluate our strategy if we don't first load the data: +
+ Click for details ```r - library(SingleCellExperiment) -#> Loading required package: SummarizedExperiment -#> Loading required package: MatrixGenerics -#> Loading required package: matrixStats -#> -#> Attaching package: 'matrixStats' -#> The following object is masked from 'package:dplyr': -#> -#> count -#> -#> Attaching package: 'MatrixGenerics' -#> The following objects are masked from 'package:matrixStats': -#> -#> colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse, -#> colCounts, colCummaxs, colCummins, colCumprods, colCumsums, -#> colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs, -#> colMads, colMaxs, colMeans2, colMedians, colMins, colOrderStats, -#> colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds, -#> colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads, -#> colWeightedMeans, colWeightedMedians, colWeightedSds, -#> colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet, -#> rowCollapse, rowCounts, rowCummaxs, rowCummins, rowCumprods, -#> rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps, -#> rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians, rowMins, -#> rowOrderStats, rowProds, rowQuantiles, rowRanges, rowRanks, -#> rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs, rowVars, -#> rowWeightedMads, rowWeightedMeans, rowWeightedMedians, -#> rowWeightedSds, rowWeightedVars -#> Loading required package: GenomicRanges -#> Loading required package: stats4 -#> Loading required package: BiocGenerics -#> -#> Attaching package: 'BiocGenerics' -#> The following objects are masked from 'package:stats': -#> -#> IQR, mad, sd, var, xtabs -#> The following objects are masked from 'package:dplyr': -#> -#> combine, intersect, setdiff, union -#> The following objects are masked from 'package:base': -#> -#> Filter, Find, Map, Position, Reduce, anyDuplicated, append, -#> as.data.frame, basename, cbind, colnames, dirname, do.call, -#> duplicated, eval, evalq, get, grep, grepl, intersect, is.unsorted, -#> lapply, mapply, match, mget, order, paste, pmax, pmax.int, pmin, -#> pmin.int, rank, rbind, rownames, sapply, setdiff, sort, table, -#> tapply, union, unique, unsplit, which.max, which.min -#> Loading required package: S4Vectors -#> -#> Attaching package: 'S4Vectors' -#> The following object is masked from 'package:tidyr': -#> -#> expand -#> The following objects are masked from 'package:dplyr': -#> -#> first, rename -#> The following objects are masked from 'package:base': -#> -#> I, expand.grid, unname -#> Loading required package: IRanges -#> -#> Attaching package: 'IRanges' -#> The following object is masked from 'package:purrr': -#> -#> reduce -#> The following objects are masked from 'package:dplyr': -#> -#> collapse, desc, slice -#> Loading required package: GenomeInfoDb -#> Loading required package: Biobase -#> Welcome to Bioconductor -#> -#> Vignettes contain introductory material; view with -#> 'browseVignettes()'. To cite Bioconductor, see -#> 'citation("Biobase")', and for packages 'citation("pkgname")'. -#> -#> Attaching package: 'Biobase' -#> The following object is masked from 'package:MatrixGenerics': -#> -#> rowMedians -#> The following objects are masked from 'package:matrixStats': -#> -#> anyMissing, rowMedians library(tidySingleCellExperiment) +``` +
-# should package this... -if (!exists("tidybarnyard")) -tidybarnyard <- tidy(readRDS(url("https://ttriche.github.io/RDS/barnyard.rds"))) -#> Warning: `tidy()` was deprecated in tidySingleCellExperiment 1.1.1. -#> tidySingleCellExperiment says: tidy() is not needed anymore. -#> This warning is displayed once every 8 hours. -#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated. + +```r +# should package this... could just instantiate from a package via data(...) +if (!exists("tidybarnyard")) { + tidybarnyard <- readRDS(url("https://ttriche.github.io/RDS/tidybarnyard.rds")) +} ``` + It will be useful to know that, when you use the `$` operator on a typical Bioconductor object (such as a _tidySingleCellExperiment_ like ours), it assumes you want to see the column data (`colData`) with that name (see below). For @@ -206,7 +81,7 @@ constructor function and it bolted them onto the side of the object. It turns out _[SingleCellExperiment](https://www.nature.com/articles/s41592-019-0654-x)_ is just a modified version of _[SummarizedExperiment](https://www.nature.com/articles/nmeth.3252)_: -![the SummarizedExperiment scheme](../figure/SummarizedExperiment.png) +![the SummarizedExperiment scheme](../figure/SummarizedExperiment.jpg) The figure from the original paper is better/easier to understand, although you can certainly read about the new one. TidySingleCellExperiment wraps the "newer" @@ -233,10 +108,10 @@ a little easier to keep track of what's going on when we subset either one. # as what is our object masquerading? show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" -#> # A SingleCellExperiment-tibble abstraction: 0 × 5 +#> # A SingleCellExperiment-tibble abstraction: 0 × 7 #> # Features=62046 | Assays=counts -#> # … with 5 variables: cell , name , experiment , method , -#> # cell +#> # … with 7 variables: cell , name , experiment , method , +#> # barcode , fracmouse , frachuman # how many rows (genes) and columns (cells) are there in our tidy barnyard? dim(tidybarnyard) @@ -285,11 +160,10 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" filter(method == "inDrops") %>% # "choose rows where method == inDrops" slice_sample %>% # "randomly slice out one row" pull("cell") -> aCell # "pull the column cell, assign to aCell" -#> Error: Can't transform a data frame with duplicate names. # your cell: show(aCell) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'show': object 'aCell' not found +#> [1] "Mixture2.inDrops.GGGAGATG-AAGAGCGT-AACCCTTG" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -299,11 +173,11 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000098847_mm10_Gm27235" +#> [1] "mm10_ENSMUSG00000027624_mm10_Epb41l1" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'j' in selecting a method for function '[': object 'aCell' not found +#> [1] 0 # note that the odds are good that you'll get a 0 for this random combination: library(Matrix) # for the `nnzero` function @@ -320,20 +194,16 @@ message(round(sparsity * 100, 1), "%") as_tibble(tidybarnyard) %>% # turn tidybarnyard's colData into a tibble slice_sample(n=100) %>% # slice out 100 random rows, and then... pull("cell") -> aHundredCells # assign the "cell" column to "aHundredCells" -#> Error: Can't transform a data frame with duplicate names. as_tibble(rowData(tidybarnyard)) %>% # turn tidybarnyard's rowData into a tibble slice_sample(n=100) %>% # slice out 100 random rows, and then pull("name") -> aHundredGenes # assign "name" column to "aHundredGenes" samples <- (length(aHundredCells) * length(aHundredGenes)) -#> Error in eval(expr, envir, enclos): object 'aHundredCells' not found nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'x' in selecting a method for function 'nnzero': error in evaluating the argument 'j' in selecting a method for function '[': object 'aHundredCells' not found sparsity_hat <- (samples - nonzero) / samples -#> Error in eval(expr, envir, enclos): object 'nonzero' not found sparsity_hat # estimated sparsity -#> Error in eval(expr, envir, enclos): object 'sparsity_hat' not found +#> [1] 0.9278 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -349,7 +219,6 @@ sample_sparsity <- function(object, cells=100, genes=100) { # the `replicate` function allows us to apply this many times over: estimates <- replicate(n=100, sample_sparsity(tidybarnyard)) # "do it 100 times" -#> Error: Can't transform a data frame with duplicate names. # plot estimates: library(ggplot2) @@ -358,7 +227,12 @@ ggplot(tibble(estimate=estimates), aes(estimate)) + geom_vline(xintercept=sparsity, color="red", lwd=3) + theme_minimal() + ggtitle("Sparsity of UMI matrix (true value in red)") -#> Error in eval_tidy(xs[[j]], mask): object 'estimates' not found +#> `stat_bin()` using `bins = 30`. Pick better value with `binwidth`. +``` + +![plot of chunk rowDataAndColumnData](figure/rowDataAndColumnData-1.png) + +```r # the Central Limit Theorem lives to fight another day, # and we have a decent idea of how to navigate our data. @@ -445,7 +319,6 @@ p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman)) + # first pass at a plot: p + ggtitle("Barnyard experiment") -#> Error: `data` must be uniquely named but has duplicate columns ``` ![plot of chunk bygenome](figure/bygenome-1.png) @@ -475,7 +348,6 @@ barnyardtibble %>% case_when(frachuman > minhuman & fracmouse < minmouse ~ "human", fracmouse > minmouse & frachuman < minhuman ~ "mouse", TRUE ~ "suspect")) -> barnyardtibble -#> Error: Can't transform a data frame with duplicate names. # add our initial stab at labeling: p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=label)) + @@ -491,7 +363,6 @@ p + geom_density2d(alpha=0.5, color="blue") + geom_vline(xintercept=minmouse, color="red") + geom_hline(yintercept=minhuman, color="red") + ggtitle("First stab at gating") -#> Error: `data` must be uniquely named but has duplicate columns ``` ![plot of chunk gating](figure/gating-1.png) @@ -512,7 +383,6 @@ barnyardtibble %>% mutate(shading = case_when(label == "suspect" ~ 0.3, doublet == TRUE ~ 0.1, TRUE ~ 0.5)) -> barnyardtibble -#> Error: Can't transform a data frame with duplicate names. # start like the previous plot, but add low and high "gates" for each species: p <- ggplot(barnyardtibble, @@ -534,7 +404,6 @@ p <- ggplot(barnyardtibble, # plot it p + ggtitle("aggregate doublet gating") -#> Error: `data` must be uniquely named but has duplicate columns ``` ![plot of chunk doubletgate](figure/doubletgate-1.png) @@ -546,7 +415,6 @@ Before we gate those out, let's see if this is related to library prep method: # reuse the plot again: p + facet_wrap(~ method) -#> Error: `data` must be uniquely named but has duplicate columns ``` ![plot of chunk byMethod](figure/byMethod-1.png) @@ -560,28 +428,22 @@ it's surprisingly less bad than many approaches ("what's fashionable?", etc). # we need a 0/1 outcome to perform logistic regression: barnyardtibble %>% mutate(classifiable = label != "suspect") -> barnyardtibble -#> Error: Can't transform a data frame with duplicate names. # logistic regression in R uses the glm() or general linear model function, # with a binomial (0/1) link, and the result can be compared like any lm(): fit0 <- glm(classifiable ~ 1, data=barnyardtibble, family=binomial) # null model -#> Error in eval(predvars, data, env): object 'classifiable' not found # add `method` to the predictors fit1 <- update(fit0, classifiable ~ method) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit0' not found # add `method` and `experiment` to the predictors fit2 <- update(fit1, classifiable ~ method + experiment) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit1' not found # add `method` and `experiment` to the predictors, no intercept fit3 <- update(fit2, classifiable ~ method + experiment + 0) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit2' not found # add `method` as the sole predictor, no intercept fit4 <- update(fit0, classifiable ~ method + 0) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fit0' not found ``` Remember that logistic regression transforms the input probabilities, and that @@ -600,21 +462,66 @@ library(coefplot) # classifiable ~ method coefplot(fit1, trans=invlogit) + theme_minimal() -#> Error in coefplot(fit1, trans = invlogit): object 'fit1' not found +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. +``` + +![plot of chunk coefficients](figure/coefficients-1.png) + +```r # classifiable ~ method + experiment coefplot(fit2, trans=invlogit) + theme_minimal() -#> Error in coefplot(fit2, trans = invlogit): object 'fit2' not found +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. +``` + +![plot of chunk coefficients](figure/coefficients-2.png) + +```r # classifiable ~ method + experiment, no intercept coefplot(fit3, trans=invlogit) + theme_minimal() -#> Error in coefplot(fit3, trans = invlogit): object 'fit3' not found +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. +``` + +![plot of chunk coefficients](figure/coefficients-3.png) + +```r # classifiable ~ method, no intercept coefplot(fit4, trans=invlogit) + theme_minimal() -#> Error in coefplot(fit4, trans = invlogit): object 'fit4' not found +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. ``` +![plot of chunk coefficients](figure/coefficients-4.png) + Any thoughts on which library prep you'd use if cost is no object? # Mixture models as an alternative to manual gating @@ -631,17 +538,10 @@ automatic gating of actual flow cytometry data this way, in fact, and it works # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) -#> __ ___________ __ _____________ -#> / |/ / ____/ / / / / / ___/_ __/ -#> / /|_/ / / / / / / / /\__ \ / / -#> / / / / /___/ /___/ /_/ /___/ // / -#> /_/ /_/\____/_____/\____//____//_/ version 5.4.8 -#> Type 'citation("mclust")' for citing this R package in publications. # since these are proportional values, it makes sense to transform them: -mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), G=1:3) -#> fitting ... -#> | | | 0% | |== | 2% | |=== | 5% | |===== | 7% | |======= | 9% | |======== | 12% | |========== | 14% | |=========== | 16% | |============= | 19% | |=============== | 21% | |================ | 23% | |================== | 26% | |==================== | 28% | |===================== | 30% | |======================= | 33% | |======================== | 35% | |========================== | 37% | |============================ | 40% | |============================= | 42% | |=============================== | 44% | |================================= | 47% | |================================== | 49% | |==================================== | 51% | |===================================== | 53% | |======================================= | 56% | |========================================= | 58% | |========================================== | 60% | |============================================ | 63% | |============================================== | 65% | |=============================================== | 67% | |================================================= | 70% | |================================================== | 72% | |==================================================== | 74% | |====================================================== | 77% | |======================================================= | 79% | |========================================================= | 81% | |=========================================================== | 84% | |============================================================ | 86% | |============================================================== | 88% | |=============================================================== | 91% | |================================================================= | 93% | |=================================================================== | 95% | |==================================================================== | 98% | |======================================================================| 100% +mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), + verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! # here I have restricted Mclust to fitting, at most, 3 components (G=1:3). # this speeds up the process and avoids some difficult questions later on ;-) @@ -649,7 +549,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), G=1:3) table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 2116 1941 142 +#> 142 1941 2116 barnyardtibble$mclass <- ifelse(mfit$classification == 2, "suspect", ifelse(mfit$classification == 1, "human", "mouse")) # not sure how do this tidily. @@ -666,7 +566,6 @@ p <- ggplot(barnyardtibble, # plot it p + ggtitle("mixture model fit") -#> Error: `data` must be uniquely named but has duplicate columns ``` ![plot of chunk mixtureModel](figure/mixtureModel-1.png) @@ -677,31 +576,59 @@ Suppose we re-run the regressions using the mixture model fits. What happens? ```r barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble -#> Error: Can't transform a data frame with duplicate names. # null model fitm0 <- glm(mclassifiable ~ 0, data=barnyardtibble, family=binomial) # random -#> Error in eval(predvars, data, env): object 'mclassifiable' not found # regress `mclassifiable` on method, no intercept: fitm1 <- update(fitm0, mclassifiable ~ method + 0) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fitm0' not found coefplot(fitm1, trans=invlogit) + theme_minimal() -#> Error in coefplot(fitm1, trans = invlogit): object 'fitm1' not found +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. +``` + +![plot of chunk remix](figure/remix-1.png) + +```r # regress `mclassifiable` on method and experiment, no intercept: fitm2 <- update(fitm0, mclassifiable ~ method + experiment + 0) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fitm0' not found coefplot(fitm2, trans=invlogit) + theme_minimal() -#> Error in coefplot(fitm2, trans = invlogit): object 'fitm2' not found +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. +``` + +![plot of chunk remix](figure/remix-2.png) + +```r # regress `mclassifiable` on method interacting with experiment, no intercept: fitm3 <- update(fitm0, mclassifiable ~ method * experiment + 0) -#> Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'update': object 'fitm0' not found coefplot(fitm3, trans=invlogit) + theme_minimal() -#> Error in coefplot(fitm3, trans = invlogit): object 'fitm3' not found +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. + +#> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please +#> use `guide = "none"` instead. ``` +![plot of chunk remix](figure/remix-3.png) + Have your thoughts on which method to use changed? Remember, each coefficient ends up being the odds that a cell can be classified, so you have to multiply through the values that are true for each cell. Given the confidence intervals @@ -713,3 +640,49 @@ One thing you might consider is to allow Mclust to use more possible values for G (the number of Groups). Its default is "up to 10". If you plot the results, do you still feel comfortable interpreting one of the groups as "suspect"? Is it still reasonable to use _glm_ (for logistic regression) in that case? + +# Sort-of-bonus: resampling + +Above, I stated that maybe we don't need thousands of cells per method. +You could always adjust the `ideal` argument to the function below to resample: +
+ Click for sample_umis() function code + +```r + +# adapted from a SingleCellExperiment-centric method for CITEseq +sample_umis <- function(umis, meta, block, ideal=300) { + + # {{{ + stopifnot(nrow(meta) == ncol(umis)) + stopifnot(length(block) == nrow(meta)) + + pops <- sort(table(block)) + samplesets <- split(seq_len(nrow(meta)), block) + + keep <- integer() + for (set in names(samplesets)) { + sset <- samplesets[[set]] + cells <- length(sset) + if (cells <= ideal) { + pct <- 100 + keep <- c(keep, sset) + message("Kept ", cells, " cells (", pct, "%) of type ", set, ".") + } else { + kept <- sample(sset, size=ideal) + pct <- round((ideal / cells) * 100) + keep <- c(keep, kept) + message("Kept ", ideal, " cells (", pct, "%) of type ", set, ".") + } + } + + pct <- round((length(keep) / ncol(umis)) * 100, 1) + message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), + " cells in ", length(samplesets), " blocks.") + umis[, keep] + # }}} + +} +``` +
+ From aa82b19ba3b2a0c9b0424a5f9ca90141ad8d34c2 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 11:03:50 -0500 Subject: [PATCH 08/25] add UMAP output and R code --- vignettes/project2_chunks/figure/UMAP.png | Bin 0 -> 83823 bytes vignettes/project2_chunks/project2_tim.R | 403 +++++++++++++++++++++ vignettes/project2_chunks/project2_tim.Rmd | 5 +- vignettes/project2_chunks/project2_tim.md | 5 +- 4 files changed, 411 insertions(+), 2 deletions(-) create mode 100644 vignettes/project2_chunks/figure/UMAP.png create mode 100644 vignettes/project2_chunks/project2_tim.R diff --git a/vignettes/project2_chunks/figure/UMAP.png b/vignettes/project2_chunks/figure/UMAP.png new file mode 100644 index 0000000000000000000000000000000000000000..090104643540e007878989cc0cdef641e8393e26 GIT binary patch literal 83823 zcmeFYb#Pt1k}qnEV~&|2c4B5`$IQ%(+sw?4Ic7U%rWj&oW@ct)W@f+bd}q%2X71d% zZ>rw?<5kT{t!*u-^;4_Wl3J}!u&lHQJPbAr2nYzgn5dvU2nhHr@O}OG0hn`&9&`W# 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0 HcmV?d00001 diff --git a/vignettes/project2_chunks/project2_tim.R b/vignettes/project2_chunks/project2_tim.R new file mode 100644 index 0000000..715db4b --- /dev/null +++ b/vignettes/project2_chunks/project2_tim.R @@ -0,0 +1,403 @@ +## ----setup, include=FALSE----------------------------------------------------- +knitr::opts_chunk$set(echo = TRUE) +knitr::opts_chunk$set(collapse = TRUE, comment = "#>") +library(devtools) +load_all("./") + + +## ---- tidysinglecell---------------------------------------------------------- + +if (!require("SingleCellExperiment")) { + BiocManager::install("SingleCellExperiment") + library(SingleCellExperiment) +} +if (!require("tidySingleCellExperiment")) { + BiocManager::install("tidySingleCellExperiment") + library(tidySingleCellExperiment) +} + + + +## ---- loadpackages------------------------------------------------------------ +library(SingleCellExperiment) +library(tidySingleCellExperiment) + + +## ---- gettidysce-------------------------------------------------------------- +# should package this... could just instantiate from a package via data(...) +if (!exists("tidybarnyard")) { + tidybarnyard <- readRDS(url("https://ttriche.github.io/RDS/tidybarnyard.rds")) +} + + +## ---- rowDataAndColumnData---------------------------------------------------- + +# as what is our object masquerading? +show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" + +# how many rows (genes) and columns (cells) are there in our tidy barnyard? +dim(tidybarnyard) + +# is this equivalent to what we get from nrow() and ncol()? +identical(dim(tidybarnyard), + c(nrow(tidybarnyard), ncol(tidybarnyard))) + +# the assay(s) should also have this many rows and columns. Do they? +identical(dim(tidybarnyard), dim(assay(tidybarnyard))) + +# there is a shortcut for the `counts` assay, since it's so common: +counts(tidybarnyard)[1:3, 1:3] + +# does each row (gene) in the object have a corresponding rowData() row? +identical(rownames(tidybarnyard), rownames(rowData(tidybarnyard))) + +# does each column (cell) in the object have a corresponding colData() row? +identical(colnames(tidybarnyard), rownames(colData(tidybarnyard))) + +# is there a rowData row and a colData row that corresponds to each UMI count? +identical(dim(counts(tidybarnyard)), + c(nrow(rowData(tidybarnyard)), nrow(colData(tidybarnyard)))) + +# does that mean we can ask for a random cell with certain attributes? +as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" + filter(method == "inDrops") %>% # "choose rows where method == inDrops" + slice_sample %>% # "randomly slice out one row" + pull("cell") -> aCell # "pull the column cell, assign to aCell" + +# your cell: +show(aCell) + +# does that mean we can ask for a random gene with certain attributes? +as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" + filter(genome == "mm10") %>% # "choose rows where genome == mm10" + slice_sample %>% # "randomly slice out one row" + pull("name") -> aGene # "pull the column name, assign to aGene" + +# your gene: +show(aGene) + +# how many copies of this random gene were found in this random cell? +counts(tidybarnyard)[aGene, aCell] + +# note that the odds are good that you'll get a 0 for this random combination: +library(Matrix) # for the `nnzero` function +sparsity <- 1 - (nnzero(counts(tidybarnyard)) / # number of nonzero counts + prod(dim(tidybarnyard))) # number of rows * columns + +# specifically, the chance of getting a zero is about 92.3%: +message(round(sparsity * 100, 1), "%") + +# This is fairly typical for a single-cell experiment, perhaps a bit low even. +# You could also take 10000 or so random samples to estimate the sparsity. +# For example, you could grab 100 genes from 100 cells at a time: +as_tibble(tidybarnyard) %>% # turn tidybarnyard's colData into a tibble + slice_sample(n=100) %>% # slice out 100 random rows, and then... + pull("cell") -> aHundredCells # assign the "cell" column to "aHundredCells" + +as_tibble(rowData(tidybarnyard)) %>% # turn tidybarnyard's rowData into a tibble + slice_sample(n=100) %>% # slice out 100 random rows, and then + pull("name") -> aHundredGenes # assign "name" column to "aHundredGenes" + +samples <- (length(aHundredCells) * length(aHundredGenes)) +nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) +sparsity_hat <- (samples - nonzero) / samples +sparsity_hat # estimated sparsity + +# In fact, we can use this scheme to look at sampling error: +sample_sparsity <- function(object, cells=100, genes=100) { + + samples <- cells * genes + columns <- pull(slice_sample(as_tibble(object), n=cells), "cell") + rows <- pull(slice_sample(as_tibble(rowData(object)), n=genes), "name") + nonzero <- nnzero(counts(object)[rows, columns]) + sparsity <- (samples - nonzero) / samples + return(sparsity) + +} + +# the `replicate` function allows us to apply this many times over: +estimates <- replicate(n=100, sample_sparsity(tidybarnyard)) # "do it 100 times" + +# plot estimates: +library(ggplot2) +ggplot(tibble(estimate=estimates), aes(estimate)) + + geom_histogram() + + geom_vline(xintercept=sparsity, color="red", lwd=3) + + theme_minimal() + + ggtitle("Sparsity of UMI matrix (true value in red)") + +# the Central Limit Theorem lives to fight another day, +# and we have a decent idea of how to navigate our data. + + +## ---- UMAP, eval=FALSE-------------------------------------------------------- +## +## library(scater) +## # it is standard to log-normalize counts +## # (although it's not actually a great idea) +## tidybarnyard <- logNormCounts(tidybarnyard) +## +## # compute UMAP embedding on the most variable genes +## tidybarnyard %>% runUMAP(ncomponents=3) -> tidyUMAP +## +## # plot using plotly, color by method +## tidyUMAP %>% +## plot_ly( +## x =~ `UMAP1`, +## y =~ `UMAP2`, +## z =~ `UMAP3`, +## color =~ method +## ) +## + + +## ---- bygenome---------------------------------------------------------------- + +# identify the mouse genes: +as_tibble(rowData(tidybarnyard)) %>% # make a tibble from the rowData + filter(genome == "mm10") %>% # select just the mouse mm10 genes + pull("name") -> mouseGenes # assign their name column to mouseGenes + +# for technical reasons, it's faster to tally expressed genes this way: +tidybarnyard$fracmouse <- + (colSums(counts(tidybarnyard)[mouseGenes, ] > 0)) / length(mouseGenes) + +# do the same thing but with human genes: +as_tibble(rowData(tidybarnyard)) %>% # make a tibble from the rowData + filter(genome == "hg19") %>% # select just the human hg19 genes + pull("name") -> humanGenes # assign their name column to humanGenes + +# same remarks as previously +tidybarnyard$frachuman <- + (colSums(counts(tidybarnyard)[humanGenes, ] > 0)) / length(humanGenes) + +# why normalize by gene count? +as_tibble(rowData(tidybarnyard)) %>% select("genome") %>% table + + +# let's use ggplot to make sense of the results: +barnyardtibble <- as_tibble(tidybarnyard) + +p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.75, color="lightblue") + + geom_density2d(alpha=0.5, color="blue") + + theme_minimal() + +# first pass at a plot: +p + ggtitle("Barnyard experiment") + + + +## ---- gating------------------------------------------------------------------ + +# arbitrarily: +minhuman <- 0.05 +minmouse <- 0.05 +# Let's label any cell that is more than 5% (0.05) of BOTH genomes as suspect. + +# add a label: +barnyardtibble %>% + mutate(label = + case_when(frachuman > minhuman & fracmouse < minmouse ~ "human", + fracmouse > minmouse & frachuman < minhuman ~ "mouse", + TRUE ~ "suspect")) -> barnyardtibble + +# add our initial stab at labeling: +p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.75) + + theme_minimal() + +# plot it with some gates and density lines: +p + geom_density2d(alpha=0.5, color="blue") + + geom_vline(xintercept=minmouse, color="red") + + geom_hline(yintercept=minhuman, color="red") + + ggtitle("First stab at gating") + + + +## ---- doubletgate------------------------------------------------------------- + +# arbitrarily: +maxhuman <- 0.21 +maxmouse <- 0.19 + +# make it obvious which cells are going to be gated out if we do this: +barnyardtibble %>% + mutate(doublet = frachuman > maxhuman | fracmouse > maxmouse) %>% + mutate(shading = case_when(label == "suspect" ~ 0.3, + doublet == TRUE ~ 0.1, + TRUE ~ 0.5)) -> barnyardtibble + +# start like the previous plot, but add low and high "gates" for each species: +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=label, alpha=I(shading))) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point() + + geom_segment(y=0, yend=minhuman, x=minmouse, xend=minmouse, color="black") + + geom_segment(y=0, yend=minhuman, x=maxmouse, xend=maxmouse, color="black") + + geom_segment(y=minhuman, yend=minhuman, x=minmouse, xend=maxmouse, + color="black") + + geom_segment(y=minhuman, yend=minhuman, x=0, xend=minmouse, color="black") + + geom_segment(y=maxhuman, yend=maxhuman, x=0, xend=minmouse, color="black") + + geom_segment(y=minhuman, yend=maxhuman, x=minmouse, xend=minmouse, + color="black") + + theme_minimal() + +# plot it +p + ggtitle("aggregate doublet gating") + + + + +## ---- byMethod---------------------------------------------------------------- + +# reuse the plot again: +p + facet_wrap(~ method) + + + +## ---- regressFactors---------------------------------------------------------- + +# we need a 0/1 outcome to perform logistic regression: +barnyardtibble %>% mutate(classifiable = label != "suspect") -> barnyardtibble + +# logistic regression in R uses the glm() or general linear model function, +# with a binomial (0/1) link, and the result can be compared like any lm(): +fit0 <- glm(classifiable ~ 1, data=barnyardtibble, family=binomial) # null model + +# add `method` to the predictors +fit1 <- update(fit0, classifiable ~ method) + +# add `method` and `experiment` to the predictors +fit2 <- update(fit1, classifiable ~ method + experiment) + +# add `method` and `experiment` to the predictors, no intercept +fit3 <- update(fit2, classifiable ~ method + experiment + 0) + +# add `method` as the sole predictor, no intercept +fit4 <- update(fit0, classifiable ~ method + 0) + + + +## ---- coefficients------------------------------------------------------------ + +# it's better to use confidence intervals than p-values for fitting purposes, +# and it's even better yet to plot them all: +library(coefplot) + +# classifiable ~ method +coefplot(fit1, trans=invlogit) + theme_minimal() + +# classifiable ~ method + experiment +coefplot(fit2, trans=invlogit) + theme_minimal() + +# classifiable ~ method + experiment, no intercept +coefplot(fit3, trans=invlogit) + theme_minimal() + +# classifiable ~ method, no intercept +coefplot(fit4, trans=invlogit) + theme_minimal() + + + +## ---- mixtureModel------------------------------------------------------------ + +# one of the greatest software packages ever written, +# fits a Gaussian mixture model with arbitrary covariance structure and uses +# a Bayesian penalization scheme to choose how many components exist in the mix +library(mclust) + +# since these are proportional values, it makes sense to transform them: +mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), + verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! +# here I have restricted Mclust to fitting, at most, 3 components (G=1:3). +# this speeds up the process and avoids some difficult questions later on ;-) + +# create a new column of the barnyard tibble with the results: +table(mfit$classification) # it turns out that we end up with less human cells +barnyardtibble$mclass <- ifelse(mfit$classification == 2, "suspect", + ifelse(mfit$classification == 1, "human", + "mouse")) # not sure how do this tidily. + +# add mixture assignments: +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.5) + + theme_minimal() + +# plot it +p + ggtitle("mixture model fit") + + + +## ---- remix------------------------------------------------------------------- + +barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble + +# null model +fitm0 <- glm(mclassifiable ~ 0, data=barnyardtibble, family=binomial) # random + +# regress `mclassifiable` on method, no intercept: +fitm1 <- update(fitm0, mclassifiable ~ method + 0) +coefplot(fitm1, trans=invlogit) + theme_minimal() + +# regress `mclassifiable` on method and experiment, no intercept: +fitm2 <- update(fitm0, mclassifiable ~ method + experiment + 0) +coefplot(fitm2, trans=invlogit) + theme_minimal() + +# regress `mclassifiable` on method interacting with experiment, no intercept: +fitm3 <- update(fitm0, mclassifiable ~ method * experiment + 0) +coefplot(fitm3, trans=invlogit) + theme_minimal() + + + +## ---- sample_umis------------------------------------------------------------- + +# adapted from a SingleCellExperiment-centric method for CITEseq +sample_umis <- function(umis, meta, block, ideal=300) { + + # {{{ + stopifnot(nrow(meta) == ncol(umis)) + stopifnot(length(block) == nrow(meta)) + + pops <- sort(table(block)) + samplesets <- split(seq_len(nrow(meta)), block) + + keep <- integer() + for (set in names(samplesets)) { + sset <- samplesets[[set]] + cells <- length(sset) + if (cells <= ideal) { + pct <- 100 + keep <- c(keep, sset) + message("Kept ", cells, " cells (", pct, "%) of type ", set, ".") + } else { + kept <- sample(sset, size=ideal) + pct <- round((ideal / cells) * 100) + keep <- c(keep, kept) + message("Kept ", ideal, " cells (", pct, "%) of type ", set, ".") + } + } + + pct <- round((length(keep) / ncol(umis)) * 100, 1) + message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), + " cells in ", length(samplesets), " blocks.") + umis[, keep] + # }}} + +} + diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index e61aec1..aeec14f 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -210,8 +210,10 @@ Often, when someone cracks open a single cell dataset, the first thing they do is to cluster the cells (based on whatever approach is fashionable). We don't need to do that here. In fact, if you do plot the usual UMAP'ed clusters, you'll mostly just see a batch effect. This takes a while to compute, so I -recommend you not bother with the next chunk. +recommend you not bother with the next chunk. I'll drop in a plotly screengrab. +
+ Only peek if you want to see why UMAP can mislead you ```{r, UMAP, eval=FALSE} library(scater) @@ -232,6 +234,7 @@ tidyUMAP %>% ) ``` +
For this project, we know that the reads from each library (cell) were competitively aligned against the mouse and human genomes. So all we really diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 36742ec..a4a439c 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -242,8 +242,10 @@ Often, when someone cracks open a single cell dataset, the first thing they do is to cluster the cells (based on whatever approach is fashionable). We don't need to do that here. In fact, if you do plot the usual UMAP'ed clusters, you'll mostly just see a batch effect. This takes a while to compute, so I -recommend you not bother with the next chunk. +recommend you not bother with the next chunk. I'll drop in a plotly screengrab. +
+ Only peek if you want to see why UMAP can mislead you ```r @@ -265,6 +267,7 @@ tidyUMAP %>% ) ``` +
For this project, we know that the reads from each library (cell) were competitively aligned against the mouse and human genomes. So all we really From 307032d83c196b2c6c24959646df23fb2afdb135 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 11:11:01 -0500 Subject: [PATCH 09/25] add UMAP hidey-ho output --- vignettes/project2_chunks/project2_tim.Rmd | 2 ++ vignettes/project2_chunks/project2_tim.md | 2 ++ 2 files changed, 4 insertions(+) diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index aeec14f..c3d2f9d 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -234,6 +234,8 @@ tidyUMAP %>% ) ``` +![UMAP plotly](figure/UMAP.png) + For this project, we know that the reads from each library (cell) were diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index a4a439c..e6b15aa 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -267,6 +267,8 @@ tidyUMAP %>% ) ``` +![UMAP 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b/vignettes/project2_chunks/project2_tim.Rmd index c3d2f9d..e54f357 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -459,9 +459,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), # create a new column of the barnyard tibble with the results: table(mfit$classification) # it turns out that we end up with less human cells -barnyardtibble$mclass <- ifelse(mfit$classification == 2, "suspect", - ifelse(mfit$classification == 1, "human", - "mouse")) # not sure how do this tidily. +barnyardtibble$mclass <- factor(mfit$classification) # add mixture assignments: p <- ggplot(barnyardtibble, @@ -482,6 +480,10 @@ Suppose we re-run the regressions using the mixture model fits. What happens? ```{r, remix} +# relabel the mixture class calls based on the previous plot +barnyardtibble %>% mutate(mclass = case_when(mclass == 1 ~ "mouse", + mclass == 3 ~ "human", + TRUE ~ "suspect"))-> barnyardtibble barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble # null model diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index e6b15aa..74354c0 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -108,10 +108,10 @@ a little easier to keep track of what's going on when we subset either one. # as what is our object masquerading? show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" -#> # A SingleCellExperiment-tibble abstraction: 0 × 7 -#> # Features=62046 | Assays=counts -#> # … with 7 variables: cell , name , experiment , method , -#> # barcode , fracmouse , frachuman +#> # A SingleCellExperiment-tibble abstraction: 0 × 8 +#> # Features=62046 | Assays=counts, logcounts +#> # … with 8 variables: cell , name , experiment , method , +#> # barcode , sizeFactor , fracmouse , frachuman # how many rows (genes) and columns (cells) are there in our tidy barnyard? dim(tidybarnyard) @@ -555,9 +555,7 @@ table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 #> 142 1941 2116 -barnyardtibble$mclass <- ifelse(mfit$classification == 2, "suspect", - ifelse(mfit$classification == 1, "human", - "mouse")) # not sure how do this tidily. +barnyardtibble$mclass <- factor(mfit$classification) # add mixture assignments: p <- ggplot(barnyardtibble, @@ -580,6 +578,10 @@ Suppose we re-run the regressions using the mixture model fits. What happens? ```r +# relabel the mixture class calls based on the previous plot +barnyardtibble %>% mutate(mclass = case_when(mclass == 1 ~ "mouse", + mclass == 3 ~ "human", + TRUE ~ "suspect"))-> barnyardtibble barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble # null model From 862f50a254486fb83ec89d3679dce9b1ce17b7ff Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 12:14:39 -0500 Subject: [PATCH 11/25] deterministically label the mixture components --- .../project2_chunks/figure/mixtureModel-1.png | Bin 31095 -> 31807 bytes vignettes/project2_chunks/figure/remix-1.png | Bin 16317 -> 16303 bytes vignettes/project2_chunks/figure/remix-2.png | Bin 18181 -> 18195 bytes vignettes/project2_chunks/figure/remix-3.png | Bin 29425 -> 29540 bytes vignettes/project2_chunks/project2_tim.R | 21 +++++++++++----- vignettes/project2_chunks/project2_tim.Rmd | 21 +++++++++++----- vignettes/project2_chunks/project2_tim.md | 23 +++++++++++++----- 7 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table(mclass, label)) +(mixlabels <- apply(tbl, 2, which.max)) + +# "whichever class isn't mostly human or mouse is suspect" +mixlabels["suspect"] <- setdiff(1:3, mixlabels[c("human","mouse")]) +mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number + +# relabel the mixture assignments: +barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble + # add mixture assignments: p <- ggplot(barnyardtibble, - aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + xlab("Mouse transcripts expressed") + scale_x_continuous(labels = scales::percent) + ylab("Human transcripts expressed") + @@ -344,11 +355,9 @@ p + ggtitle("mixture model fit") ## ---- remix------------------------------------------------------------------- -# relabel the mixture class calls based on the previous plot -barnyardtibble %>% mutate(mclass = case_when(mclass == 1 ~ "mouse", - mclass == 3 ~ "human", - TRUE ~ "suspect"))-> barnyardtibble -barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble +# classifiable _by mixture model_ +barnyardtibble %>% + mutate(mclassifiable = mixlabel != "suspect") -> barnyardtibble # null model fitm0 <- glm(mclassifiable ~ 0, data=barnyardtibble, family=binomial) # random diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index e54f357..d935845 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -461,9 +461,20 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells barnyardtibble$mclass <- factor(mfit$classification) +# confusion matrix helps us assign correspondence +tbl <- with(barnyardtibble, table(mclass, label)) +(mixlabels <- apply(tbl, 2, which.max)) + +# "whichever class isn't mostly human or mouse is suspect" +mixlabels["suspect"] <- setdiff(1:3, mixlabels[c("human","mouse")]) +mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number + +# relabel the mixture assignments: +barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble + # add mixture assignments: p <- ggplot(barnyardtibble, - aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + xlab("Mouse transcripts expressed") + scale_x_continuous(labels = scales::percent) + ylab("Human transcripts expressed") + @@ -480,11 +491,9 @@ Suppose we re-run the regressions using the mixture model fits. What happens? ```{r, remix} -# relabel the mixture class calls based on the previous plot -barnyardtibble %>% mutate(mclass = case_when(mclass == 1 ~ "mouse", - mclass == 3 ~ "human", - TRUE ~ "suspect"))-> barnyardtibble -barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble +# classifiable _by mixture model_ +barnyardtibble %>% + mutate(mclassifiable = mixlabel != "suspect") -> barnyardtibble # null model fitm0 <- glm(mclassifiable ~ 0, data=barnyardtibble, family=binomial) # random diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 74354c0..13450ec 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -557,9 +557,22 @@ table(mfit$classification) # it turns out that we end up with less human cells #> 142 1941 2116 barnyardtibble$mclass <- factor(mfit$classification) +# confusion matrix helps us assign correspondence +tbl <- with(barnyardtibble, table(mclass, label)) +(mixlabels <- apply(tbl, 2, which.max)) +#> human mouse suspect +#> 3 2 2 + +# "whichever class isn't mostly human or mouse is suspect" +mixlabels["suspect"] <- setdiff(1:3, mixlabels[c("human","mouse")]) +mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number + +# relabel the mixture assignments: +barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble + # add mixture assignments: p <- ggplot(barnyardtibble, - aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + xlab("Mouse transcripts expressed") + scale_x_continuous(labels = scales::percent) + ylab("Human transcripts expressed") + @@ -578,11 +591,9 @@ Suppose we re-run the regressions using the mixture model fits. What happens? ```r -# relabel the mixture class calls based on the previous plot -barnyardtibble %>% mutate(mclass = case_when(mclass == 1 ~ "mouse", - mclass == 3 ~ "human", - TRUE ~ "suspect"))-> barnyardtibble -barnyardtibble %>% mutate(mclassifiable = mclass != "suspect") -> barnyardtibble +# classifiable _by mixture model_ +barnyardtibble %>% + mutate(mclassifiable = mixlabel != "suspect") -> barnyardtibble # null model fitm0 <- glm(mclassifiable ~ 0, data=barnyardtibble, family=binomial) # random From 1a93d34b8b60aba85ac163827044de920f1febf0 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 12:21:26 -0500 Subject: [PATCH 12/25] rerun mixture labeling from scratch... --- .../project2_chunks/figure/mixlabels-1.png | Bin 0 -> 32597 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31807 -> 31095 bytes vignettes/project2_chunks/project2_tim.R | 24 ++++++- vignettes/project2_chunks/project2_tim.Rmd | 27 +++++++- vignettes/project2_chunks/project2_tim.md | 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zu9CQiA-(1r+MnCL{dPlmNT|gBU9JPKvVGtY0Du~g9z6oVEB0R(Vc?J#@{SMyO+u)E zC+J_$xxoiehJgVC)#ErS@h>m*b>FiMd$mw@*xT7{|NUDqi4NZih;1gHd+7O7j?bIq(N95_30Dgl>hol1fkHxayBsa`v<&eL4$+Snjhpb zFfmO7?mR1IeZU+A4AbsFdH`suKr5)OU6-H#8_>m|O3>4m0oUj8i_-@m2GH17iwFJ} zfYxH??0n(f2K+htV|0;+KBxgdyw=^XEl=NV;Dz^SXlcBO-oD5 z-(SpH?Z+zUh7L|nNmf@tKF`j^mRI5_t5N^q!0*7P*?PFT6{x6yj`Bgn0P?c}EBu&b za@L)iTu{&jV6~rIEi5c7`JRj*+y+xntcHM*C2DPL?E%9Pg!VL>?hU~Cf(tx21%rHp zkcemzgg+o#UC%VMKVTz4BwAFL0HH1LT6P5UFaSE-tyA3#0V;(2(|7cMRR$o205)lv zha>@n$!>1dPCZQt2?^k;0CH6L#k(XgZ{wNxRpi6N0b6HY@E@P>S`!iyKKO!wDTLRg z8YG86{O=43N=;1#gB2V+;e;=PxVY);o=uh?vG@b?cCOYgadvn5^PrAR>n}ZxEXq1y zk6R3~{B(i+2i@@*Fbs_7OiXkACh(vh0dZ)0yGEj6!id(74tmjlvz=e?&4|~HMjZM{ to=+E<*b+l5kpF*%q%8QqXGE7&IX@2Sjj?@Gfax}*D61;-RSFjT{{V0D^E3be diff --git a/vignettes/project2_chunks/project2_tim.R b/vignettes/project2_chunks/project2_tim.R index cd7946c..36f7514 100644 --- a/vignettes/project2_chunks/project2_tim.R +++ b/vignettes/project2_chunks/project2_tim.R @@ -327,6 +327,23 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells barnyardtibble$mclass <- factor(mfit$classification) +# plot the results +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.5) + + theme_minimal() + +# plot it +p + ggtitle("mixture model fit") + + + +## ---- mixlabels--------------------------------------------------------------- + # confusion matrix helps us assign correspondence tbl <- with(barnyardtibble, table(mclass, label)) (mixlabels <- apply(tbl, 2, which.max)) @@ -338,7 +355,10 @@ mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number # relabel the mixture assignments: barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble -# add mixture assignments: +# how did we do? +with(barnyardtibble, kable(mixlabel, label)) + +# add mixture labels to the plot: p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + xlab("Mouse transcripts expressed") + @@ -349,7 +369,7 @@ p <- ggplot(barnyardtibble, theme_minimal() # plot it -p + ggtitle("mixture model fit") +p + ggtitle("mixture model fit with labels") diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index d935845..3fc4db1 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -437,6 +437,7 @@ coefplot(fit4, trans=invlogit) + theme_minimal() Any thoughts on which library prep you'd use if cost is no object? + # Mixture models as an alternative to manual gating Incidentally, we could also use a mixture model to classify all the points, and @@ -461,6 +462,25 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells barnyardtibble$mclass <- factor(mfit$classification) +# plot the results +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.5) + + theme_minimal() + +# plot it +p + ggtitle("mixture model fit") + +``` + +It looks like we will need to match up the mixture classes with our labels. + +```{r, mixlabels} + # confusion matrix helps us assign correspondence tbl <- with(barnyardtibble, table(mclass, label)) (mixlabels <- apply(tbl, 2, which.max)) @@ -472,7 +492,10 @@ mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number # relabel the mixture assignments: barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble -# add mixture assignments: +# how did we do? +with(barnyardtibble, kable(mixlabel, label)) + +# add mixture labels to the plot: p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + xlab("Mouse transcripts expressed") + @@ -483,7 +506,7 @@ p <- ggplot(barnyardtibble, theme_minimal() # plot it -p + ggtitle("mixture model fit") +p + ggtitle("mixture model fit with labels") ``` diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 13450ec..574e189 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -108,10 +108,10 @@ a little easier to keep track of what's going on when we subset either one. # as what is our object masquerading? show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" -#> # A SingleCellExperiment-tibble abstraction: 0 × 8 -#> # Features=62046 | Assays=counts, logcounts -#> # … with 8 variables: cell , name , experiment , method , -#> # barcode , sizeFactor , fracmouse , frachuman +#> # A SingleCellExperiment-tibble abstraction: 0 × 5 +#> # Features=62046 | Assays=counts +#> # … with 5 variables: cell , name , experiment , method , +#> # barcode # how many rows (genes) and columns (cells) are there in our tidy barnyard? dim(tidybarnyard) @@ -467,6 +467,19 @@ library(coefplot) # classifiable ~ method coefplot(fit1, trans=invlogit) + theme_minimal() +#> Warning: `funs()` was deprecated in dplyr 0.8.0. +#> Please use a list of either functions or lambdas: +#> +#> # Simple named list: +#> list(mean = mean, median = median) +#> +#> # Auto named with `tibble::lst()`: +#> tibble::lst(mean, median) +#> +#> # Using lambdas +#> list(~ mean(., trim = .2), ~ median(., na.rm = TRUE)) +#> This warning is displayed once every 8 hours. +#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated. #> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please #> use `guide = "none"` instead. @@ -529,6 +542,7 @@ coefplot(fit4, trans=invlogit) + theme_minimal() Any thoughts on which library prep you'd use if cost is no object? + # Mixture models as an alternative to manual gating Incidentally, we could also use a mixture model to classify all the points, and @@ -543,6 +557,12 @@ automatic gating of actual flow cytometry data this way, in fact, and it works # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) +#> __ ___________ __ _____________ +#> / |/ / ____/ / / / / / ___/_ __/ +#> / /|_/ / / / / / / / /\__ \ / / +#> / / / / /___/ /___/ /_/ /___/ // / +#> /_/ /_/\____/_____/\____//____//_/ version 5.4.8 +#> Type 'citation("mclust")' for citing this R package in publications. # since these are proportional values, it makes sense to transform them: mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), @@ -557,6 +577,27 @@ table(mfit$classification) # it turns out that we end up with less human cells #> 142 1941 2116 barnyardtibble$mclass <- factor(mfit$classification) +# plot the results +p <- ggplot(barnyardtibble, + aes(x=fracmouse, y=frachuman, color=mclass, shape=label)) + + xlab("Mouse transcripts expressed") + + scale_x_continuous(labels = scales::percent) + + ylab("Human transcripts expressed") + + scale_y_continuous(labels = scales::percent) + + geom_point(alpha=0.5) + + theme_minimal() + +# plot it +p + ggtitle("mixture model fit") +``` + +![plot of chunk mixtureModel](figure/mixtureModel-1.png) + +It looks like we will need to match up the mixture classes with our labels. + + +```r + # confusion matrix helps us assign correspondence tbl <- with(barnyardtibble, table(mclass, label)) (mixlabels <- apply(tbl, 2, which.max)) @@ -570,7 +611,11 @@ mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number # relabel the mixture assignments: barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble -# add mixture assignments: +# how did we do? +with(barnyardtibble, kable(mixlabel, label)) +#> Error in switch(format, pandoc = "simple", markdown = "pipe", format): EXPR must be a length 1 vector + +# add mixture labels to the plot: p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + xlab("Mouse transcripts expressed") + @@ -581,10 +626,10 @@ p <- ggplot(barnyardtibble, theme_minimal() # plot it -p + ggtitle("mixture model fit") +p + ggtitle("mixture model fit with labels") ``` -![plot of chunk mixtureModel](figure/mixtureModel-1.png) +![plot of chunk mixlabels](figure/mixlabels-1.png) Suppose we re-run the regressions using the mixture model fits. What happens? From 87f6bcc272b53cc2778618a284dd0398fcdf01ed Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 12:23:54 -0500 Subject: [PATCH 13/25] remove kable for table --- vignettes/project2_chunks/project2_tim.R | 7 ++-- vignettes/project2_chunks/project2_tim.Rmd | 10 ++++-- vignettes/project2_chunks/project2_tim.md | 39 +++++++++------------- 3 files changed, 28 insertions(+), 28 deletions(-) diff --git a/vignettes/project2_chunks/project2_tim.R b/vignettes/project2_chunks/project2_tim.R index 36f7514..2d69345 100644 --- a/vignettes/project2_chunks/project2_tim.R +++ b/vignettes/project2_chunks/project2_tim.R @@ -310,13 +310,16 @@ coefplot(fit4, trans=invlogit) + theme_minimal() -## ---- mixtureModel------------------------------------------------------------ +## ---- loadmclust-------------------------------------------------------------- # one of the greatest software packages ever written, # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) + + +## ---- mixtureModel------------------------------------------------------------ # since these are proportional values, it makes sense to transform them: mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! @@ -356,7 +359,7 @@ mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble # how did we do? -with(barnyardtibble, kable(mixlabel, label)) +with(barnyardtibble, table(mixlabel, label)) # add mixture labels to the plot: p <- ggplot(barnyardtibble, diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 3fc4db1..0ab3556 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -445,13 +445,19 @@ in fact that is how I'd usually do it. One of my lab members has been working on automatic gating of actual flow cytometry data this way, in fact, and it works *great*. So without further ado... -```{r, mixtureModel} +

+ load mclust +```{r, loadmclust} # one of the greatest software packages ever written, # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) +``` +
+ +```{r, mixtureModel} # since these are proportional values, it makes sense to transform them: mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! @@ -493,7 +499,7 @@ mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble # how did we do? -with(barnyardtibble, kable(mixlabel, label)) +with(barnyardtibble, table(mixlabel, label)) # add mixture labels to the plot: p <- ggplot(barnyardtibble, diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 574e189..54934da 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -108,10 +108,10 @@ a little easier to keep track of what's going on when we subset either one. # as what is our object masquerading? show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" -#> # A SingleCellExperiment-tibble abstraction: 0 × 5 +#> # A SingleCellExperiment-tibble abstraction: 0 × 7 #> # Features=62046 | Assays=counts -#> # … with 5 variables: cell , name , experiment , method , -#> # barcode +#> # … with 7 variables: cell , name , experiment , method , +#> # barcode , fracmouse , frachuman # how many rows (genes) and columns (cells) are there in our tidy barnyard? dim(tidybarnyard) @@ -467,19 +467,6 @@ library(coefplot) # classifiable ~ method coefplot(fit1, trans=invlogit) + theme_minimal() -#> Warning: `funs()` was deprecated in dplyr 0.8.0. -#> Please use a list of either functions or lambdas: -#> -#> # Simple named list: -#> list(mean = mean, median = median) -#> -#> # Auto named with `tibble::lst()`: -#> tibble::lst(mean, median) -#> -#> # Using lambdas -#> list(~ mean(., trim = .2), ~ median(., na.rm = TRUE)) -#> This warning is displayed once every 8 hours. -#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated. #> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please #> use `guide = "none"` instead. @@ -550,6 +537,8 @@ in fact that is how I'd usually do it. One of my lab members has been working on automatic gating of actual flow cytometry data this way, in fact, and it works *great*. So without further ado... +
+ load mclust ```r @@ -557,13 +546,11 @@ automatic gating of actual flow cytometry data this way, in fact, and it works # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) -#> __ ___________ __ _____________ -#> / |/ / ____/ / / / / / ___/_ __/ -#> / /|_/ / / / / / / / /\__ \ / / -#> / / / / /___/ /___/ /_/ /___/ // / -#> /_/ /_/\____/_____/\____//____//_/ version 5.4.8 -#> Type 'citation("mclust")' for citing this R package in publications. +``` +
+ +```r # since these are proportional values, it makes sense to transform them: mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! @@ -612,8 +599,12 @@ mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble # how did we do? -with(barnyardtibble, kable(mixlabel, label)) -#> Error in switch(format, pandoc = "simple", markdown = "pipe", format): EXPR must be a length 1 vector +with(barnyardtibble, table(mixlabel, label)) +#> label +#> mixlabel human mouse suspect +#> human 18 5 119 +#> mouse 0 1716 225 +#> suspect 1925 0 191 # add mixture labels to the plot: p <- ggplot(barnyardtibble, From 81e1898d4c3578bcec5b34a12ccd5c68c1909da9 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Wed, 17 Nov 2021 12:32:13 -0500 Subject: [PATCH 14/25] brute-force label the mixture components --- .../figure/mixlabeledplot-1.png | Bin 0 -> 32988 bytes 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with(barnyardtibble, table(mixlabel, label)) +# specifically, do we label all the human and mouse cells confidently? +with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] + + +## ---- mixlabeledplot---------------------------------------------------------- + # add mixture labels to the plot: p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 0ab3556..4ec132c 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -489,18 +489,27 @@ It looks like we will need to match up the mixture classes with our labels. # confusion matrix helps us assign correspondence tbl <- with(barnyardtibble, table(mclass, label)) -(mixlabels <- apply(tbl, 2, which.max)) - -# "whichever class isn't mostly human or mouse is suspect" -mixlabels["suspect"] <- setdiff(1:3, mixlabels[c("human","mouse")]) -mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number +mouseclass <- which.max(tbl[, "mouse"]) +humanclass <- which.max(tbl[, "human"]) # relabel the mixture assignments: -barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble +barnyardtibble %>% + mutate(mixlabel = case_when(mclass == mouseclass ~ "mouse", + mclass == humanclass ~ "human", + TRUE ~ "suspect") + ) -> barnyardtibble # how did we do? with(barnyardtibble, table(mixlabel, label)) +# specifically, do we label all the human and mouse cells confidently? +with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] +``` + +How did we do? + +```{r, mixlabeledplot} + # add mixture labels to the plot: p <- ggplot(barnyardtibble, aes(x=fracmouse, y=frachuman, color=mixlabel, shape=label)) + diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 54934da..8f940c6 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -587,24 +587,37 @@ It looks like we will need to match up the mixture classes with our labels. # confusion matrix helps us assign correspondence tbl <- with(barnyardtibble, table(mclass, label)) -(mixlabels <- apply(tbl, 2, which.max)) -#> human mouse suspect -#> 3 2 2 - -# "whichever class isn't mostly human or mouse is suspect" -mixlabels["suspect"] <- setdiff(1:3, mixlabels[c("human","mouse")]) -mixnames <- names(mixlabels)[c(1, 2, 3)] # label by number +mouseclass <- which.max(tbl[, "mouse"]) +humanclass <- which.max(tbl[, "human"]) # relabel the mixture assignments: -barnyardtibble %>% mutate(mixlabel = mixnames[mclass]) -> barnyardtibble +barnyardtibble %>% + mutate(mixlabel = case_when(mclass == mouseclass ~ "mouse", + mclass == humanclass ~ "human", + TRUE ~ "suspect") + ) -> barnyardtibble # how did we do? with(barnyardtibble, table(mixlabel, label)) #> label #> mixlabel human mouse suspect -#> human 18 5 119 +#> human 1925 0 191 #> mouse 0 1716 225 -#> suspect 1925 0 191 +#> suspect 18 5 119 + +# specifically, do we label all the human and mouse cells confidently? +with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] +#> label +#> mixlabel human mouse +#> human 1925 0 +#> mouse 0 1716 +#> suspect 18 5 +``` + +How did we do? + + +```r # add mixture labels to the plot: p <- ggplot(barnyardtibble, @@ -620,7 +633,7 @@ p <- ggplot(barnyardtibble, p + ggtitle("mixture model fit with labels") ``` -![plot of chunk mixlabels](figure/mixlabels-1.png) +![plot of chunk mixlabeledplot](figure/mixlabeledplot-1.png) Suppose we re-run the regressions using the mixture model fits. What happens? 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.../project2_chunks/figure/coefficients-1.png | Bin 16336 -> 16366 bytes .../project2_chunks/figure/coefficients-2.png | Bin 17947 -> 17926 bytes .../project2_chunks/figure/coefficients-3.png | Bin 18024 -> 18024 bytes .../project2_chunks/figure/coefficients-4.png | Bin 16536 -> 16540 bytes .../project2_chunks/figure/competitive-1.png | Bin 0 -> 13153 bytes .../figure/confidenceintervals-1.png | Bin 0 -> 16101 bytes .../figure/mixlabeledplot-1.png | Bin 32988 -> 32978 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31095 -> 31465 bytes vignettes/project2_chunks/figure/remix-1.png | Bin 16379 -> 16342 bytes vignettes/project2_chunks/figure/remix-2.png | Bin 18106 -> 18136 bytes vignettes/project2_chunks/figure/remix-3.png | Bin 29430 -> 29458 bytes .../figure/rowDataAndColumnData-1.png | Bin 10320 -> 9864 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 0 -> 26529 bytes vignettes/project2_chunks/project2_tim.R | 144 +++++- vignettes/project2_chunks/project2_tim.Rmd | 253 ++++++++++- vignettes/project2_chunks/project2_tim.md | 409 +++++++++++++++++- 19 files changed, 772 insertions(+), 39 deletions(-) create mode 100644 vignettes/project2_chunks/figure/CITEseq.png create mode 100644 vignettes/project2_chunks/figure/competitive-1.png create mode 100644 vignettes/project2_chunks/figure/confidenceintervals-1.png create mode 100644 vignettes/project2_chunks/figure/sinaplot-1.png diff --git a/DESCRIPTION b/DESCRIPTION index 238fb6e..b95f3ae 100644 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -11,8 +11,8 @@ Description: For the practical sessions in Experimental Design & Biostats 2021, Rstudio to \url{https://rpubs.com/}, so all that's left is to make sure that dependencies for assignments are in place. This package also includes a few convenience functions (phelp, lsos, etc.). -Version: 0.11.16 -Date: 2021-11-16 +Version: 0.11.20 +Date: 2021-11-20 Authors@R: person("Triche", "Lab", role=c("aut", "cre"), email="trichelab@gmail.com") Author: Triche Lab [aut, cre] Maintainer: Triche Lab @@ -23,6 +23,7 @@ Depends: R (>= 4.0.0), conflicted, ggforce, + gtools, googlesheets4, moderndive, tidyverse, diff --git a/vignettes/project2_chunks/figure/CITEseq.png b/vignettes/project2_chunks/figure/CITEseq.png new file mode 100644 index 0000000000000000000000000000000000000000..d5742b66320f07cc0ba1e9232ce9efd94897517c GIT binary patch literal 161481 zcmdqJhd&$X{ACUDP(U^S;;QRNJeHxSy>q&JJ}>jHc5n3Mnov1VT8<( zy}jSxeLv6hzJJ4ee?Ir!-Edvk?>vw5IKJz=0@YL$>8KA-lSm{wrSozcBoetOiA0t_ zMTtMz$f{JsFBA?kN}5#oj~kUq0DiyUQC`RKvW=Oevyr_i>E>-4Ytxes#`dPBw;jxF z9H+>uBuFGyl9HUXrc1&^qO+T(d}p1fvvd1BKHoFD?kYV}VWqz-B~7uFfzf3v#nv=O z`ta%VtzYI+X&|^73N8ydooaPfSdZNZzcPS33T0p&ncg<-Y6`rF?Y9 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z0-kuF&)eDCL*SP-WCs?Vvv;dk3+uqxNZR+yPqc3wxeeiP{YWd6KkWWs}Ky3JM@r>NfgIvdWX z3Zn>l_KNE>F_?=#!c7@Eh&xD)f2#wj`6+fo02mgYXgu^Z+2$ z!fyx&^e4fDa$#PCh4)@UYLxZX?Ps;!+D%`fA{0C{Sp(>k@wP30SW6!~cy8_^i@3?h zr=hB9P~(~jYa=9t`P!>sW;OO(tAKUd2__?8x6^L;uH&-;`(B54))b3V^&|={0;t=; z-o8wiCpBU6P z6BQE!>%;-**3?5yZ|{0P#98-13IaXD-t0Q)i|`uNd&8`Ni>aZhXc1{S4C7lto&;q0`$0aSBm% slice %>% mutate + result=if_else(mixlabel == "suspect", "failure", "success")) + tbl <- table(select(subs, method, result)) # force of habit + probs <- as(sweep(tbl, 1, rowSums(tbl), `/`), "matrix") + rownames(probs) <- NULL # avoid hassles when stacking + tibble(cbind(method=rownames(tbl), data.frame(probs))) + +} + +# a simple bootstrap (for efficiency, we don't bother grabbing the UMIs at all) +# per the above (and to make computing intervals easier), we'll use 200x200 +subsamples <- replicate(n=200, simplify=FALSE, # to make it easier to iterate + sample_umis(tidybarnyard, as_tibble(tidybarnyard), + tidybarnyard$method, ideal=200, + justnames=TRUE, verbose=FALSE)) +names(subsamples) <- paste0("run", seq_along(subsamples)) + +# purrr::map maps a function over a list +library(purrr) + +# evaluate performance across 100 bootstrap samples of 100 cells per method +runs <- purrr::map(subsamples, performance_by_method, atibble=barnyardtibble) + +# for plotting, stack them +results <- bind_rows(runs) + + + +## ---- sinaplot---------------------------------------------------------------- + +# fire up geom_sina because it rules: +library(ggforce) + +# for better resolution, you could resample batches of 500, 1000, ... +results %>% +# mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% + ggplot(aes(method, success, color=method)) + + scale_y_continuous(labels = scales::label_percent()) + + ylab("classification performance") + + geom_sina(show.legend=FALSE) + + coord_flip() + + theme_minimal() + + ggtitle("Cell classification results by prep, 200 cells apiece") + + + +## ---- confidenceintervals----------------------------------------------------- + +# ground rules +CI <- 0.95 # must be between 0 and 1 +lower <- (1 - CI) / 2 # defined by the value of CI +upper <- 1 - lower # defined by the value of lower +middle <- (lower + upper) / 2 # this is a tautology and a test +plot_title <- paste0(CI * 100, "% empirical confidence intervals, resampled") + +# grouping +results %>% + group_by(method) %>% # create a grouped data frame + summarize(lower=quantile(success, lower), # lower limit of the CI + middle=quantile(success, middle), # middle (== median) + upper=quantile(success, upper)) -> # upper limit of the CI + CIs # assign to a new tibble, "CIs" + +# reorder to plot +library(forcats) +CIs %>% + mutate(method = fct_reorder(method, desc(middle))) %>% + ggplot(aes(x=method, ymin=lower, y=middle, ymax=upper, color=method)) + + scale_y_continuous(labels = scales::label_percent()) + + geom_pointrange(show.legend=FALSE) + + ylab("Classification success") + + coord_flip() + + theme_minimal() + + ggtitle(plot_title) + + + +## ---- competitive------------------------------------------------------------- + +# assign 1 point to the winner, 0 otherwise +# in the case of ties, split the point N ways +score_methods <- function(run) { + + top <- max(run$success) + run$points <- as.numeric(run$success == top) + score <- run$points / sum(run$points) + names(score) <- run$method + return(score) } +# tally up the scores +runs %>% + purrr::map(score_methods) %>% # map the function score_methods onto each run + bind_rows() %>% # bind the results as rows of a tibble + colSums -> scores # compute the column sums and assign to scores + +# tibble-ify for plotting +outcome <- tibble(method=names(scores), + score=scores, + scheme="unblocked") + +# plot it +outcome %>% ggplot(aes(x=scheme, y=score, fill=method)) + + geom_col(position="fill") + + theme_minimal() + + ggtitle("Winning method, by resampling scheme") + + diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 4ec132c..16504e0 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -15,7 +15,7 @@ vignette: > knitr::opts_chunk$set(echo = TRUE) knitr::opts_chunk$set(collapse = TRUE, comment = "#>") library(devtools) -load_all("./") +#load_all("./") ``` # Background @@ -52,6 +52,10 @@ if (!require("tidySingleCellExperiment")) { BiocManager::install("tidySingleCellExperiment") library(tidySingleCellExperiment) } +if (!require("tidyverse")) { + BiocManager::install("tidyverse") + library(tidyverse) +} ``` @@ -458,6 +462,11 @@ library(mclust) ```{r, mixtureModel} + +# `logit` is from the `gtools` package: +library(gtools) +# Note to self: always compile vignettes in a fresh session with R --vanilla :-/ + # since these are proportional values, it makes sense to transform them: mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! @@ -571,9 +580,8 @@ You could always adjust the `ideal` argument to the function below to resample: ```{r, sample_umis} # adapted from a SingleCellExperiment-centric method for CITEseq -sample_umis <- function(umis, meta, block, ideal=300) { +sample_umis <- function(umis, meta, block, ideal=300, verbose=TRUE, justnames=FALSE) { - # {{{ stopifnot(nrow(meta) == ncol(umis)) stopifnot(length(block) == nrow(meta)) @@ -587,22 +595,249 @@ sample_umis <- function(umis, meta, block, ideal=300) { if (cells <= ideal) { pct <- 100 keep <- c(keep, sset) - message("Kept ", cells, " cells (", pct, "%) of type ", set, ".") + if (verbose) message("Kept ",cells," cells (",pct,"%) of type ",set,".") } else { kept <- sample(sset, size=ideal) pct <- round((ideal / cells) * 100) keep <- c(keep, kept) - message("Kept ", ideal, " cells (", pct, "%) of type ", set, ".") + if (verbose) message("Kept ",ideal," cells (",pct,"%) of type ",set,".") } } pct <- round((length(keep) / ncol(umis)) * 100, 1) - message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), - " cells in ", length(samplesets), " blocks.") - umis[, keep] - # }}} + if (verbose) { + message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), + " cells in ", length(samplesets), " blocks.") + } + + if (justnames) return(keep) + else return(umis[, keep]) } ``` +Let's suppose we decided that we didn't care which mixture the cells came from, +and we just wanted to calibrate our coefficient estimates for each prep method. +Recall the `replicate` loop earlier for estimating sparsity. If we take, say, +200 samples of 200 cells per method, what do our estimates (i.e., no intercept, +purely marginal) look like? The function above works just fine on a tidysce, and +since we are not fitting an intercept, we can perform a simplified version of +logistic regression simply by computing marginals. + +```{r, resampletidysce} + +# simple classifier: is it suspect? then it's a failure +performance_by_method <- function(subsample, atibble) { + + subs <- mutate(slice(atibble, subsample), # i.e., tbl %>% slice %>% mutate + result=if_else(mixlabel == "suspect", "failure", "success")) + tbl <- table(select(subs, method, result)) # force of habit + probs <- as(sweep(tbl, 1, rowSums(tbl), `/`), "matrix") + rownames(probs) <- NULL # avoid hassles when stacking + tibble(cbind(method=rownames(tbl), data.frame(probs))) + +} + +# a simple bootstrap (for efficiency, we don't bother grabbing the UMIs at all) +# per the above (and to make computing intervals easier), we'll use 200x200 +subsamples <- replicate(n=200, simplify=FALSE, # to make it easier to iterate + sample_umis(tidybarnyard, as_tibble(tidybarnyard), + tidybarnyard$method, ideal=200, + justnames=TRUE, verbose=FALSE)) +names(subsamples) <- paste0("run", seq_along(subsamples)) + +# purrr::map maps a function over a list +library(purrr) + +# evaluate performance across 100 bootstrap samples of 100 cells per method +runs <- purrr::map(subsamples, performance_by_method, atibble=barnyardtibble) + +# for plotting, stack them +results <- bind_rows(runs) + +``` + +Per usual, it helps to plot the results. + +```{r, sinaplot} + +# fire up geom_sina because it rules: +library(ggforce) + +# for better resolution, you could resample batches of 500, 1000, ... +results %>% +# mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% + ggplot(aes(method, success, color=method)) + + scale_y_continuous(labels = scales::label_percent()) + + ylab("classification performance") + + geom_sina(show.legend=FALSE) + + coord_flip() + + theme_minimal() + + ggtitle("Cell classification results by prep, 200 cells apiece") + +``` + +_Question:_ I commented out a line in the plot above. Would it make it clearer? + +You might conclude that we could directly compute confidence intervals from the +above results. You would be correct. Let's do this in a tidy-ish fashion: + +```{r, confidenceintervals} + +# ground rules +CI <- 0.95 # must be between 0 and 1 +lower <- (1 - CI) / 2 # defined by the value of CI +upper <- 1 - lower # defined by the value of lower +middle <- (lower + upper) / 2 # this is a tautology and a test +plot_title <- paste0(CI * 100, "% empirical confidence intervals, resampled") + +# grouping +results %>% + group_by(method) %>% # create a grouped data frame + summarize(lower=quantile(success, lower), # lower limit of the CI + middle=quantile(success, middle), # middle (== median) + upper=quantile(success, upper)) -> # upper limit of the CI + CIs # assign to a new tibble, "CIs" + +# reorder to plot +library(forcats) +CIs %>% + mutate(method = fct_reorder(method, desc(middle))) %>% + ggplot(aes(x=method, ymin=lower, y=middle, ymax=upper, color=method)) + + scale_y_continuous(labels = scales::label_percent()) + + geom_pointrange(show.legend=FALSE) + + ylab("Classification success") + + coord_flip() + + theme_minimal() + + ggtitle(plot_title) + +``` + +_Question (not required)_: Can you overlay the two summaries above? (I can't.) + +_Question (not required)_: Is this also feasible for each combination of +(mixture, method)? If so, how would you structure your resampling scheme? + +Perhaps you've previously been told that it is not possible to exclude the +possibility that two groups (or groups of samples) are equivalent at the 5% +alpha level if their 95% confidence intervals overlap. This is in fact the case, +since inverting a test at a given alpha is equivalent to computing a (1-alpha)% +confidence interval. Congratulations, you just computed all marginal comparisons +for these methods at a significance level of 0.05. + +_Question:_ What happens if you take bigger samples, or more of them? + +Suppose we went back to the original dataset (or, for the sake of argument, a +larger dataset) and resampled the hell out of it. At some point, do you suppose +you could choose a single "best" library prep method with 95% confidence (i.e., +a family-wise error rate of 0.05, or 5%)? This is roughly equivalent to a Holm +(or, if you can't count, Bonferroni) correction a the same alpha level. + +_Question:_ Did you have to make any assumptions to do this? Which ones? + +If you think this is a discussion about statistical power and significance, +you're right. If you think that resampling is always the answer... + +_Question:_ Is there a limit to how tight your intervals can be from resampling? + +(Hint: Yes. Consider the number of possible unique outcomes and permutations. +Note that if we want exact CIs, we need to avoid double-counting the results.) + +_Question:_ Can you come up with a resampling based scheme that is exhaustive, +i.e., it samples every possible permutation of draws from the data, no more, no +less? Does this scheme change if you block on multiple variables or groupings? + +(Hint: Look up "leave-one-out cross-validation".) + + +# Tests and other impedimentia + +You can also ask more interesting questions, like how many times a given method +ends up with the best performance out of the lot in each sampling run. This is +perhaps a better representation of what to expect if you applied a prep, or a +labeling scheme, or a test across multiple smaller experiments. Like, say, if +your instructor asked you to determine a sample size at which you had an 80% +chance of successfully discriminating some rare cell type from others, and oh +by the way, you need to choose between protocols that cost different amounts +per cell. And perhaps you wanted a way to look at different classifiers as well +as different prep or sort methods for your Extremely Important Rare Cell Type. +Because as you can probably guess from the above, you're not limited to just +comparing methods, or even using the same sample size per block, to do this. + +```{r, competitive} + +# assign 1 point to the winner, 0 otherwise +# in the case of ties, split the point N ways +score_methods <- function(run) { + + top <- max(run$success) + run$points <- as.numeric(run$success == top) + score <- run$points / sum(run$points) + names(score) <- run$method + return(score) + +} + +# tally up the scores +runs %>% + purrr::map(score_methods) %>% # map the function score_methods onto each run + bind_rows() %>% # bind the results as rows of a tibble + colSums -> scores # compute the column sums and assign to scores + +# tibble-ify for plotting +outcome <- tibble(method=names(scores), + score=scores, + scheme="unblocked") + +# plot it +outcome %>% ggplot(aes(x=scheme, y=score, fill=method)) + + geom_col(position="fill") + + theme_minimal() + + ggtitle("Winning method, by resampling scheme") + +``` + +So that's why I left the resampling in. Because it is in fact the state of the +art for single-cell experimental design evaluation in the context of power and +efficiency. (You want the best balance of false positives and false negatives +possible for your experiment, and can choose whatever tools you wish to achieve +that balance; this is the point of a well executed experimental design.) You +will note that nowhere in the above resampling schemes have we assumed much of +anything other than that a mixture model is usually optimal for cell labeling +(because it is, in the general case; though you should feel free to investigate +whether something else might work better for any specific experiment of yours). + +# Philosophical bloviation + +You might begin to think that experimental design is about navigating tradeoffs. +One tradeoff that most people (including myself) are rarely equipped to navigate +by intuition is what test or method to use. So don't: let the data tell you. And +absolutely do include some positive and negative controls in your experiments, +whether they are _in vitro_, _in vivo_, or _in silico_. This is particularly +important when evaluating new and/or fashionable techniques: + +[The state of the art in single-cell batch correction benchmarking](https://www.biorxiv.org/content/10.1101/2021.11.15.468733v1.full) + +_Question:_ Can you think of a way to include some negative controls in this +experiment? We know that there are empty droplets in many methods, and we know +that doublets are not uncommon in droplet-based methods. Suppose you had access +to an independent label or labels on each cell, as you might find in a +[CITE-seq experiment](https://www.nature.com/articles/s41592-020-01050-x/figures/1). If you haven't bumped into one before, here's roughly how it works: + +![a CITE-seq experiment](figure/CITEseq.png) + +Specifically, suppose you coded up the human and mouse cells (or the cell types +you actually cared about) with batch (HTO, hash-tag oligo) labels, like in the +'S' column of the matrix above. (Think of it as a great big tibble, and think +of the pieces of the big matrix as alternative assays.) Further, the protein +tags would help quite a bit if you think there really are expressed differences +within the usual cell subtypes (perhaps the ones you labeled with the hashtags). + +_Question_: Now can you think of a way to devise positive and negative controls +that doesn't depend upon consensus or what anyone else thinks? + +Congratulations, now you're even a little beyond the state of the published art. +Apologies if Scott or I gave you the impression that [most published research is +false](https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.0020124). That might be discouraging, and we wouldn't want to suggest that there +is [anything wrong with academic research](https://royalsocietypublishing.org/doi/10.1098/rsos.160384)! diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 8f940c6..886e782 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -44,10 +44,112 @@ if (!require("SingleCellExperiment")) { BiocManager::install("SingleCellExperiment") library(SingleCellExperiment) } +#> Loading required package: SingleCellExperiment +#> Loading required package: SummarizedExperiment +#> Loading required package: MatrixGenerics +#> Loading required package: matrixStats +#> +#> Attaching package: 'MatrixGenerics' +#> The following objects are masked from 'package:matrixStats': +#> +#> colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse, +#> colCounts, colCummaxs, colCummins, colCumprods, colCumsums, +#> colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs, +#> colMads, colMaxs, colMeans2, colMedians, colMins, colOrderStats, +#> colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds, +#> colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads, +#> colWeightedMeans, colWeightedMedians, colWeightedSds, +#> colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet, +#> rowCollapse, rowCounts, rowCummaxs, rowCummins, rowCumprods, +#> rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps, +#> rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians, rowMins, +#> rowOrderStats, rowProds, rowQuantiles, rowRanges, rowRanks, +#> rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs, rowVars, +#> rowWeightedMads, rowWeightedMeans, rowWeightedMedians, +#> rowWeightedSds, rowWeightedVars +#> Loading required package: GenomicRanges +#> Loading required package: stats4 +#> Loading required package: BiocGenerics +#> +#> Attaching package: 'BiocGenerics' +#> The following objects are masked from 'package:stats': +#> +#> IQR, mad, sd, var, xtabs +#> The following objects are masked from 'package:base': +#> +#> anyDuplicated, append, as.data.frame, basename, cbind, colnames, +#> dirname, do.call, duplicated, eval, evalq, Filter, Find, get, grep, +#> grepl, intersect, is.unsorted, lapply, Map, mapply, match, mget, +#> order, paste, pmax, pmax.int, pmin, pmin.int, Position, rank, +#> rbind, Reduce, rownames, sapply, setdiff, sort, table, tapply, +#> union, unique, unsplit, which.max, which.min +#> Loading required package: S4Vectors +#> +#> Attaching package: 'S4Vectors' +#> The following objects are masked from 'package:base': +#> +#> expand.grid, I, unname +#> Loading required package: IRanges +#> Loading required package: GenomeInfoDb +#> Loading required package: Biobase +#> Welcome to Bioconductor +#> +#> Vignettes contain introductory material; view with +#> 'browseVignettes()'. To cite Bioconductor, see +#> 'citation("Biobase")', and for packages 'citation("pkgname")'. +#> +#> Attaching package: 'Biobase' +#> The following object is masked from 'package:MatrixGenerics': +#> +#> rowMedians +#> The following objects are masked from 'package:matrixStats': +#> +#> anyMissing, rowMedians if (!require("tidySingleCellExperiment")) { BiocManager::install("tidySingleCellExperiment") library(tidySingleCellExperiment) } +#> Loading required package: tidySingleCellExperiment +#> +#> Attaching package: 'tidySingleCellExperiment' +#> The following object is masked from 'package:IRanges': +#> +#> slice +#> The following object is masked from 'package:S4Vectors': +#> +#> rename +#> The following object is masked from 'package:matrixStats': +#> +#> count +#> The following object is masked from 'package:stats': +#> +#> filter +if (!require("tidyverse")) { + BiocManager::install("tidyverse") + library(tidyverse) +} +#> Loading required package: tidyverse +#> ── Attaching packages ─────────────────────────────────────── tidyverse 1.3.1 ── +#> ✔ ggplot2 3.3.5 ✔ purrr 0.3.4 +#> ✔ tibble 3.1.6 ✔ dplyr 1.0.7 +#> ✔ tidyr 1.1.4 ✔ stringr 1.4.0 +#> ✔ readr 2.1.0 ✔ forcats 0.5.1 +#> ── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ── +#> ✖ dplyr::add_count() masks tidySingleCellExperiment::add_count() +#> ✖ dplyr::bind_cols() masks tidySingleCellExperiment::bind_cols() +#> ✖ dplyr::bind_rows() masks tidySingleCellExperiment::bind_rows() +#> ✖ dplyr::collapse() masks IRanges::collapse() +#> ✖ dplyr::combine() masks Biobase::combine(), BiocGenerics::combine() +#> ✖ dplyr::count() masks tidySingleCellExperiment::count(), matrixStats::count() +#> ✖ dplyr::desc() masks IRanges::desc() +#> ✖ tidyr::expand() masks S4Vectors::expand() +#> ✖ dplyr::filter() masks tidySingleCellExperiment::filter(), stats::filter() +#> ✖ dplyr::first() masks S4Vectors::first() +#> ✖ dplyr::lag() masks stats::lag() +#> ✖ ggplot2::Position() masks BiocGenerics::Position(), base::Position() +#> ✖ purrr::reduce() masks GenomicRanges::reduce(), IRanges::reduce() +#> ✖ dplyr::rename() masks tidySingleCellExperiment::rename(), S4Vectors::rename() +#> ✖ dplyr::slice() masks tidySingleCellExperiment::slice(), IRanges::slice() ``` @@ -108,10 +210,10 @@ a little easier to keep track of what's going on when we subset either one. # as what is our object masquerading? show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" -#> # A SingleCellExperiment-tibble abstraction: 0 × 7 +#> # A SingleCellExperiment-tibble abstraction: 0 × 5 #> # Features=62046 | Assays=counts -#> # … with 7 variables: cell , name , experiment , method , -#> # barcode , fracmouse , frachuman +#> # … with 5 variables: cell , name , experiment , method , +#> # barcode # how many rows (genes) and columns (cells) are there in our tidy barnyard? dim(tidybarnyard) @@ -163,7 +265,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture2.inDrops.GGGAGATG-AAGAGCGT-AACCCTTG" +#> [1] "Mixture1.inDrops.ATGCGGAG-TATCCTCT-GATTTACC" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -173,7 +275,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000027624_mm10_Epb41l1" +#> [1] "mm10_ENSMUSG00000095493_mm10_A630023A22Rik" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] @@ -181,6 +283,14 @@ counts(tidybarnyard)[aGene, aCell] # note that the odds are good that you'll get a 0 for this random combination: library(Matrix) # for the `nnzero` function +#> +#> Attaching package: 'Matrix' +#> The following objects are masked from 'package:tidyr': +#> +#> expand, pack, unpack +#> The following object is masked from 'package:S4Vectors': +#> +#> expand sparsity <- 1 - (nnzero(counts(tidybarnyard)) / # number of nonzero counts prod(dim(tidybarnyard))) # number of rows * columns @@ -203,7 +313,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9278 +#> [1] 0.9239 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -467,6 +577,19 @@ library(coefplot) # classifiable ~ method coefplot(fit1, trans=invlogit) + theme_minimal() +#> Warning: `funs()` was deprecated in dplyr 0.8.0. +#> Please use a list of either functions or lambdas: +#> +#> # Simple named list: +#> list(mean = mean, median = median) +#> +#> # Auto named with `tibble::lst()`: +#> tibble::lst(mean, median) +#> +#> # Using lambdas +#> list(~ mean(., trim = .2), ~ median(., na.rm = TRUE)) +#> This warning is displayed once every 8 hours. +#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated. #> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please #> use `guide = "none"` instead. @@ -546,11 +669,27 @@ automatic gating of actual flow cytometry data this way, in fact, and it works # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) +#> __ ___________ __ _____________ +#> / |/ / ____/ / / / / / ___/_ __/ +#> / /|_/ / / / / / / / /\__ \ / / +#> / / / / /___/ /___/ /_/ /___/ // / +#> /_/ /_/\____/_____/\____//____//_/ version 5.4.8 +#> Type 'citation("mclust")' for citing this R package in publications. +#> +#> Attaching package: 'mclust' +#> The following object is masked from 'package:purrr': +#> +#> map ``` ```r + +# `logit` is from the `gtools` package: +library(gtools) +# Note to self: always compile vignettes in a fresh session with R --vanilla :-/ + # since these are proportional values, it makes sense to transform them: mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), verbose=FALSE, G=1:3) # verbose=FALSE to avoid progress bar! @@ -561,7 +700,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 142 1941 2116 +#> 2124 1941 134 barnyardtibble$mclass <- factor(mfit$classification) # plot the results @@ -601,17 +740,17 @@ barnyardtibble %>% with(barnyardtibble, table(mixlabel, label)) #> label #> mixlabel human mouse suspect -#> human 1925 0 191 +#> human 1927 0 197 #> mouse 0 1716 225 -#> suspect 18 5 119 +#> suspect 16 5 113 # specifically, do we label all the human and mouse cells confidently? with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] #> label #> mixlabel human mouse -#> human 1925 0 +#> human 1927 0 #> mouse 0 1716 -#> suspect 18 5 +#> suspect 16 5 ``` How did we do? @@ -718,9 +857,8 @@ You could always adjust the `ideal` argument to the function below to resample: ```r # adapted from a SingleCellExperiment-centric method for CITEseq -sample_umis <- function(umis, meta, block, ideal=300) { +sample_umis <- function(umis, meta, block, ideal=300, verbose=TRUE, justnames=FALSE) { - # {{{ stopifnot(nrow(meta) == ncol(umis)) stopifnot(length(block) == nrow(meta)) @@ -734,22 +872,255 @@ sample_umis <- function(umis, meta, block, ideal=300) { if (cells <= ideal) { pct <- 100 keep <- c(keep, sset) - message("Kept ", cells, " cells (", pct, "%) of type ", set, ".") + if (verbose) message("Kept ",cells," cells (",pct,"%) of type ",set,".") } else { kept <- sample(sset, size=ideal) pct <- round((ideal / cells) * 100) keep <- c(keep, kept) - message("Kept ", ideal, " cells (", pct, "%) of type ", set, ".") + if (verbose) message("Kept ",ideal," cells (",pct,"%) of type ",set,".") } } pct <- round((length(keep) / ncol(umis)) * 100, 1) - message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), - " cells in ", length(samplesets), " blocks.") - umis[, keep] - # }}} + if (verbose) { + message("Kept ", length(keep), " (", pct, "%) of ", ncol(umis), + " cells in ", length(samplesets), " blocks.") + } + + if (justnames) return(keep) + else return(umis[, keep]) } ``` +Let's suppose we decided that we didn't care which mixture the cells came from, +and we just wanted to calibrate our coefficient estimates for each prep method. +Recall the `replicate` loop earlier for estimating sparsity. If we take, say, +200 samples of 200 cells per method, what do our estimates (i.e., no intercept, +purely marginal) look like? The function above works just fine on a tidysce, and +since we are not fitting an intercept, we can perform a simplified version of +logistic regression simply by computing marginals. + + +```r + +# simple classifier: is it suspect? then it's a failure +performance_by_method <- function(subsample, atibble) { + + subs <- mutate(slice(atibble, subsample), # i.e., tbl %>% slice %>% mutate + result=if_else(mixlabel == "suspect", "failure", "success")) + tbl <- table(select(subs, method, result)) # force of habit + probs <- as(sweep(tbl, 1, rowSums(tbl), `/`), "matrix") + rownames(probs) <- NULL # avoid hassles when stacking + tibble(cbind(method=rownames(tbl), data.frame(probs))) + +} + +# a simple bootstrap (for efficiency, we don't bother grabbing the UMIs at all) +# per the above (and to make computing intervals easier), we'll use 200x200 +subsamples <- replicate(n=200, simplify=FALSE, # to make it easier to iterate + sample_umis(tidybarnyard, as_tibble(tidybarnyard), + tidybarnyard$method, ideal=200, + justnames=TRUE, verbose=FALSE)) +names(subsamples) <- paste0("run", seq_along(subsamples)) + +# purrr::map maps a function over a list +library(purrr) + +# evaluate performance across 100 bootstrap samples of 100 cells per method +runs <- purrr::map(subsamples, performance_by_method, atibble=barnyardtibble) + +# for plotting, stack them +results <- bind_rows(runs) +``` + +Per usual, it helps to plot the results. + + +```r + +# fire up geom_sina because it rules: +library(ggforce) + +# for better resolution, you could resample batches of 500, 1000, ... +results %>% +# mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% + ggplot(aes(method, success, color=method)) + + scale_y_continuous(labels = scales::label_percent()) + + ylab("classification performance") + + geom_sina(show.legend=FALSE) + + coord_flip() + + theme_minimal() + + ggtitle("Cell classification results by prep, 200 cells apiece") +``` + +![plot of chunk sinaplot](figure/sinaplot-1.png) + +_Question:_ I commented out a line in the plot above. Would it make it clearer? + +You might conclude that we could directly compute confidence intervals from the +above results. You would be correct. Let's do this in a tidy-ish fashion: + + +```r + +# ground rules +CI <- 0.95 # must be between 0 and 1 +lower <- (1 - CI) / 2 # defined by the value of CI +upper <- 1 - lower # defined by the value of lower +middle <- (lower + upper) / 2 # this is a tautology and a test +plot_title <- paste0(CI * 100, "% empirical confidence intervals, resampled") + +# grouping +results %>% + group_by(method) %>% # create a grouped data frame + summarize(lower=quantile(success, lower), # lower limit of the CI + middle=quantile(success, middle), # middle (== median) + upper=quantile(success, upper)) -> # upper limit of the CI + CIs # assign to a new tibble, "CIs" + +# reorder to plot +library(forcats) +CIs %>% + mutate(method = fct_reorder(method, desc(middle))) %>% + ggplot(aes(x=method, ymin=lower, y=middle, ymax=upper, color=method)) + + scale_y_continuous(labels = scales::label_percent()) + + geom_pointrange(show.legend=FALSE) + + ylab("Classification success") + + coord_flip() + + theme_minimal() + + ggtitle(plot_title) +``` + +![plot of chunk confidenceintervals](figure/confidenceintervals-1.png) + +_Question (not required)_: Can you overlay the two summaries above? (I can't.) + +_Question (not required)_: Is this also feasible for each combination of +(mixture, method)? If so, how would you structure your resampling scheme? + +Perhaps you've previously been told that it is not possible to exclude the +possibility that two groups (or groups of samples) are equivalent at the 5% +alpha level if their 95% confidence intervals overlap. This is in fact the case, +since inverting a test at a given alpha is equivalent to computing a (1-alpha)% +confidence interval. Congratulations, you just computed all marginal comparisons +for these methods at a significance level of 0.05. + +_Question:_ What happens if you take bigger samples, or more of them? + +Suppose we went back to the original dataset (or, for the sake of argument, a +larger dataset) and resampled the hell out of it. At some point, do you suppose +you could choose a single "best" library prep method with 95% confidence (i.e., +a family-wise error rate of 0.05, or 5%)? This is roughly equivalent to a Holm +(or, if you can't count, Bonferroni) correction a the same alpha level. + +_Question:_ Did you have to make any assumptions to do this? Which ones? + +If you think this is a discussion about statistical power and significance, +you're right. If you think that resampling is always the answer... + +_Question:_ Is there a limit to how tight your intervals can be from resampling? + +(Hint: Yes. Consider the number of possible unique outcomes and permutations. +Note that if we want exact CIs, we need to avoid double-counting the results.) + +_Question:_ Can you come up with a resampling based scheme that is exhaustive, +i.e., it samples every possible permutation of draws from the data, no more, no +less? Does this scheme change if you block on multiple variables or groupings? + +(Hint: Look up "leave-one-out cross-validation".) + + +# Tests and other impedimentia + +You can also ask more interesting questions, like how many times a given method +ends up with the best performance out of the lot in each sampling run. This is +perhaps a better representation of what to expect if you applied a prep, or a +labeling scheme, or a test across multiple smaller experiments. Like, say, if +your instructor asked you to determine a sample size at which you had an 80% +chance of successfully discriminating some rare cell type from others, and oh +by the way, you need to choose between protocols that cost different amounts +per cell. And perhaps you wanted a way to look at different classifiers as well +as different prep or sort methods for your Extremely Important Rare Cell Type. +Because as you can probably guess from the above, you're not limited to just +comparing methods, or even using the same sample size per block, to do this. + + +```r + +# assign 1 point to the winner, 0 otherwise +# in the case of ties, split the point N ways +score_methods <- function(run) { + + top <- max(run$success) + run$points <- as.numeric(run$success == top) + score <- run$points / sum(run$points) + names(score) <- run$method + return(score) + +} + +# tally up the scores +runs %>% + purrr::map(score_methods) %>% # map the function score_methods onto each run + bind_rows() %>% # bind the results as rows of a tibble + colSums -> scores # compute the column sums and assign to scores + +# tibble-ify for plotting +outcome <- tibble(method=names(scores), + score=scores, + scheme="unblocked") + +# plot it +outcome %>% ggplot(aes(x=scheme, y=score, fill=method)) + + geom_col(position="fill") + + theme_minimal() + + ggtitle("Winning method, by resampling scheme") +``` + +![plot of chunk competitive](figure/competitive-1.png) + +So that's why I left the resampling in. Because it is in fact the state of the +art for single-cell experimental design evaluation in the context of power and +efficiency. (You want the best balance of false positives and false negatives +possible for your experiment, and can choose whatever tools you wish to achieve +that balance; this is the point of a well executed experimental design.) You +will note that nowhere in the above resampling schemes have we assumed much of +anything other than that a mixture model is usually optimal for cell labeling +(because it is, in the general case; though you should feel free to investigate +whether something else might work better for any specific experiment of yours). + +# Philosophical bloviation + +You might begin to think that experimental design is about navigating tradeoffs. +One tradeoff that most people (including myself) are rarely equipped to navigate +by intuition is what test or method to use. So don't: let the data tell you. And +absolutely do include some positive and negative controls in your experiments, +whether they are _in vitro_, _in vivo_, or _in silico_. This is particularly +important when evaluating new and/or fashionable techniques: + +[The state of the art in single-cell batch correction benchmarking](https://www.biorxiv.org/content/10.1101/2021.11.15.468733v1.full) + +_Question:_ Can you think of a way to include some negative controls in this +experiment? We know that there are empty droplets in many methods, and we know +that doublets are not uncommon in droplet-based methods. Suppose you had access +to an independent label or labels on each cell, as you might find in a +[CITE-seq experiment](https://www.nature.com/articles/s41592-020-01050-x/figures/1). If you haven't bumped into one before, here's roughly how it works: + +![a CITE-seq experiment](figure/CITEseq.png) + +Specifically, suppose you coded up the human and mouse cells (or the cell types +you actually cared about) with batch (HTO, hash-tag oligo) labels, like in the +'S' column of the matrix above. (Think of it as a great big tibble, and think +of the pieces of the big matrix as alternative assays.) Further, the protein +tags would help quite a bit if you think there really are expressed differences +within the usual cell subtypes (perhaps the ones you labeled with the hashtags). + +_Question_: Now can you think of a way to devise positive and negative controls +that doesn't depend upon consensus or what anyone else thinks? + +Congratulations, now you're even a little beyond the state of the published art. +Apologies if Scott or I gave you the impression that [most published research is +false](https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.0020124). That might be discouraging, and we wouldn't want to suggest that there +is [anything wrong with academic research](https://royalsocietypublishing.org/doi/10.1098/rsos.160384)! From 37d0b774a934a24aefa3d1fc3737b094dd2c0946 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 16:02:12 -0500 Subject: [PATCH 17/25] Address @svad8's issue with tidySingleCellExperiment --- .../project2_chunks/figure/competitive-1.png | Bin 13153 -> 13131 bytes .../figure/confidenceintervals-1.png | Bin 16101 -> 15295 bytes .../figure/mixlabeledplot-1.png | Bin 32978 -> 32988 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31465 -> 31475 bytes vignettes/project2_chunks/figure/remix-1.png | Bin 16342 -> 16374 bytes vignettes/project2_chunks/figure/remix-2.png | Bin 18136 -> 18079 bytes vignettes/project2_chunks/figure/remix-3.png | Bin 29458 -> 29440 bytes .../figure/rowDataAndColumnData-1.png | Bin 9864 -> 10455 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 26529 -> 26640 bytes vignettes/project2_chunks/project2_tim.R | 11 ++ vignettes/project2_chunks/project2_tim.Rmd | 18 +- vignettes/project2_chunks/project2_tim.md | 171 +++--------------- 12 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rowWeightedVars -#> Loading required package: GenomicRanges -#> Loading required package: stats4 -#> Loading required package: BiocGenerics -#> -#> Attaching package: 'BiocGenerics' -#> The following objects are masked from 'package:stats': -#> -#> IQR, mad, sd, var, xtabs -#> The following objects are masked from 'package:base': -#> -#> anyDuplicated, append, as.data.frame, basename, cbind, colnames, -#> dirname, do.call, duplicated, eval, evalq, Filter, Find, get, grep, -#> grepl, intersect, is.unsorted, lapply, Map, mapply, match, mget, -#> order, paste, pmax, pmax.int, pmin, pmin.int, Position, rank, -#> rbind, Reduce, rownames, sapply, setdiff, sort, table, tapply, -#> union, unique, unsplit, which.max, which.min -#> Loading required package: S4Vectors -#> -#> Attaching package: 'S4Vectors' -#> The following objects are masked from 'package:base': -#> -#> expand.grid, I, unname -#> Loading required package: IRanges -#> Loading required package: GenomeInfoDb -#> Loading required package: Biobase -#> Welcome to Bioconductor -#> -#> Vignettes contain introductory material; view with -#> 'browseVignettes()'. To cite Bioconductor, see -#> 'citation("Biobase")', and for packages 'citation("pkgname")'. -#> -#> Attaching package: 'Biobase' -#> The following object is masked from 'package:MatrixGenerics': -#> -#> rowMedians -#> The following objects are masked from 'package:matrixStats': -#> -#> anyMissing, rowMedians if (!require("tidySingleCellExperiment")) { BiocManager::install("tidySingleCellExperiment") library(tidySingleCellExperiment) } -#> Loading required package: tidySingleCellExperiment -#> -#> Attaching package: 'tidySingleCellExperiment' -#> The following object is masked from 'package:IRanges': -#> -#> slice -#> The following object is masked from 'package:S4Vectors': -#> -#> rename -#> The following object is masked from 'package:matrixStats': -#> -#> count -#> The following object is masked from 'package:stats': -#> -#> filter if (!require("tidyverse")) { BiocManager::install("tidyverse") library(tidyverse) } -#> Loading required package: tidyverse -#> ── Attaching packages ─────────────────────────────────────── tidyverse 1.3.1 ── -#> ✔ ggplot2 3.3.5 ✔ purrr 0.3.4 -#> ✔ tibble 3.1.6 ✔ dplyr 1.0.7 -#> ✔ tidyr 1.1.4 ✔ stringr 1.4.0 -#> ✔ readr 2.1.0 ✔ forcats 0.5.1 -#> ── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ── -#> ✖ dplyr::add_count() masks tidySingleCellExperiment::add_count() -#> ✖ dplyr::bind_cols() masks tidySingleCellExperiment::bind_cols() -#> ✖ dplyr::bind_rows() masks tidySingleCellExperiment::bind_rows() -#> ✖ dplyr::collapse() masks IRanges::collapse() -#> ✖ dplyr::combine() masks Biobase::combine(), BiocGenerics::combine() -#> ✖ dplyr::count() masks tidySingleCellExperiment::count(), matrixStats::count() -#> ✖ dplyr::desc() masks IRanges::desc() -#> ✖ tidyr::expand() masks S4Vectors::expand() -#> ✖ dplyr::filter() masks tidySingleCellExperiment::filter(), stats::filter() -#> ✖ dplyr::first() masks S4Vectors::first() -#> ✖ dplyr::lag() masks stats::lag() -#> ✖ ggplot2::Position() masks BiocGenerics::Position(), base::Position() -#> ✖ purrr::reduce() masks GenomicRanges::reduce(), IRanges::reduce() -#> ✖ dplyr::rename() masks tidySingleCellExperiment::rename(), S4Vectors::rename() -#> ✖ dplyr::slice() masks tidySingleCellExperiment::slice(), IRanges::slice() ``` @@ -201,8 +103,23 @@ us some tools to stay in the Tidyverse as much as possible while working with single-cell data. (It turns out that the underlying data makes this somewhat less trivial than you might expect. Fortunately, you don't have to care.) -Concretely, let's see how these moving parts fit together. The `dim` function -provides dimensions for a rectangular object, rows x columns, which makes it +There is a [little annoyance in _tidySingleCellExperiment_](https://github.com/stemangiola/tidySingleCellExperiment/issues/38) that we need to side-step to move along. Just to be sure, let's fix this and make sure we don't hit it later on: + + +```r + +# if any column in `tidybarnyard` column data is named `cell`, rename it +# this is currently a bug in tidySingleCellExperiment: +# https://github.com/stemangiola/tidySingleCellExperiment/issues/38 +# +names(colData(tidybarnyard)) <- sub("cell", # pattern + "barcode", # replacement + names(colData(tidybarnyard))) # strings +``` + +Now, let's see how these moving parts in the figure fit together, from the +ground up. We will make use of the `dim` function, which provides dimensions +for a rectangular object in the form rows x columns. This will also make it a little easier to keep track of what's going on when we subset either one. @@ -210,10 +127,10 @@ a little easier to keep track of what's going on when we subset either one. # as what is our object masquerading? show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" -#> # A SingleCellExperiment-tibble abstraction: 0 × 5 +#> # A SingleCellExperiment-tibble abstraction: 0 × 7 #> # Features=62046 | Assays=counts -#> # … with 5 variables: cell , name , experiment , method , -#> # barcode +#> # … with 7 variables: cell , name , experiment , method , +#> # barcode , fracmouse , frachuman # how many rows (genes) and columns (cells) are there in our tidy barnyard? dim(tidybarnyard) @@ -265,7 +182,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture1.inDrops.ATGCGGAG-TATCCTCT-GATTTACC" +#> [1] "Mixture1.inDrops.CAGATGGG-TATCCTCT-AGTTTAGA" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -275,7 +192,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000095493_mm10_A630023A22Rik" +#> [1] "mm10_ENSMUSG00000034648_mm10_Lrrn1" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] @@ -283,14 +200,6 @@ counts(tidybarnyard)[aGene, aCell] # note that the odds are good that you'll get a 0 for this random combination: library(Matrix) # for the `nnzero` function -#> -#> Attaching package: 'Matrix' -#> The following objects are masked from 'package:tidyr': -#> -#> expand, pack, unpack -#> The following object is masked from 'package:S4Vectors': -#> -#> expand sparsity <- 1 - (nnzero(counts(tidybarnyard)) / # number of nonzero counts prod(dim(tidybarnyard))) # number of rows * columns @@ -313,7 +222,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9239 +#> [1] 0.937 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -577,19 +486,6 @@ library(coefplot) # classifiable ~ method coefplot(fit1, trans=invlogit) + theme_minimal() -#> Warning: `funs()` was deprecated in dplyr 0.8.0. -#> Please use a list of either functions or lambdas: -#> -#> # Simple named list: -#> list(mean = mean, median = median) -#> -#> # Auto named with `tibble::lst()`: -#> tibble::lst(mean, median) -#> -#> # Using lambdas -#> list(~ mean(., trim = .2), ~ median(., na.rm = TRUE)) -#> This warning is displayed once every 8 hours. -#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated. #> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please #> use `guide = "none"` instead. @@ -669,17 +565,6 @@ automatic gating of actual flow cytometry data this way, in fact, and it works # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) -#> __ ___________ __ _____________ -#> / |/ / ____/ / / / / / ___/_ __/ -#> / /|_/ / / / / / / / /\__ \ / / -#> / / / / /___/ /___/ /_/ /___/ // / -#> /_/ /_/\____/_____/\____//____//_/ version 5.4.8 -#> Type 'citation("mclust")' for citing this R package in publications. -#> -#> Attaching package: 'mclust' -#> The following object is masked from 'package:purrr': -#> -#> map ``` @@ -700,7 +585,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 2124 1941 134 +#> 2116 1941 142 barnyardtibble$mclass <- factor(mfit$classification) # plot the results @@ -740,17 +625,17 @@ barnyardtibble %>% with(barnyardtibble, table(mixlabel, label)) #> label #> mixlabel human mouse suspect -#> human 1927 0 197 +#> human 1925 0 191 #> mouse 0 1716 225 -#> suspect 16 5 113 +#> suspect 18 5 119 # specifically, do we label all the human and mouse cells confidently? with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] #> label #> mixlabel human mouse -#> human 1927 0 +#> human 1925 0 #> mouse 0 1716 -#> suspect 16 5 +#> suspect 18 5 ``` How did we do? From 1e2632060bf4f1052bbe8abab3e40f50f4b63765 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 16:09:55 -0500 Subject: [PATCH 18/25] clear up the text and links towards the end --- .../project2_chunks/figure/competitive-1.png | Bin 13131 -> 13147 bytes .../figure/confidenceintervals-1.png | Bin 15295 -> 15832 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31475 -> 32191 bytes .../figure/rowDataAndColumnData-1.png | Bin 10455 -> 9857 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 26640 -> 26006 bytes vignettes/project2_chunks/project2_tim.Rmd | 11 ++++------ vignettes/project2_chunks/project2_tim.md | 19 ++++++++---------- 7 files changed, 12 insertions(+), 18 deletions(-) diff --git a/vignettes/project2_chunks/figure/competitive-1.png b/vignettes/project2_chunks/figure/competitive-1.png index e34b8a3908cc4302cafa0ead4991c100ffdc6be9..10568053d517c42983245ce62f9db56dfbd8e9fa 100644 GIT binary patch literal 13147 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edb81ed..e1f2808 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -829,9 +829,7 @@ One tradeoff that most people (including myself) are rarely equipped to navigate by intuition is what test or method to use. So don't: let the data tell you. And absolutely do include some positive and negative controls in your experiments, whether they are _in vitro_, _in vivo_, or _in silico_. This is particularly -important when evaluating new and/or fashionable techniques: - -[The state of the art in single-cell batch correction benchmarking](https://www.biorxiv.org/content/10.1101/2021.11.15.468733v1.full) +important when [evaluating new and/or fashionable techniques](https://www.biorxiv.org/content/10.1101/2021.11.15.468733v1.full). _Question:_ Can you think of a way to include some negative controls in this experiment? We know that there are empty droplets in many methods, and we know @@ -845,11 +843,10 @@ Specifically, suppose you coded up the human and mouse cells (or the cell types you actually cared about) with batch (HTO, hash-tag oligo) labels, like in the 'S' column of the matrix above. (Think of it as a great big tibble, and think of the pieces of the big matrix as alternative assays.) Further, the protein -tags would help quite a bit if you think there really are expressed differences -within the usual cell subtypes (perhaps the ones you labeled with the hashtags). +tags could help quite a bit if you think there really are expressed differences +within the usual cell subtypes. -_Question_: Now can you think of a way to devise positive and negative controls -that doesn't depend upon consensus or what anyone else thinks? +_Question_: Can you use this to devise positive and negative controls in a way that makes biological sense? Congratulations, now you're even a little beyond the state of the published art. Apologies if Scott or I gave you the impression that [most published research is diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 656fc77..85fa849 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -182,7 +182,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture1.inDrops.CAGATGGG-TATCCTCT-AGTTTAGA" +#> [1] "Mixture2.inDrops.CTGAGCGT-AGAATGCG-CCTACTAG" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -192,7 +192,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000034648_mm10_Lrrn1" +#> [1] "mm10_ENSMUSG00000045268_mm10_Zfp691" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] @@ -222,7 +222,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.937 +#> [1] 0.9225 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -585,7 +585,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 2116 1941 142 +#> 1941 2116 142 barnyardtibble$mclass <- factor(mfit$classification) # plot the results @@ -983,9 +983,7 @@ One tradeoff that most people (including myself) are rarely equipped to navigate by intuition is what test or method to use. So don't: let the data tell you. And absolutely do include some positive and negative controls in your experiments, whether they are _in vitro_, _in vivo_, or _in silico_. This is particularly -important when evaluating new and/or fashionable techniques: - -[The state of the art in single-cell batch correction benchmarking](https://www.biorxiv.org/content/10.1101/2021.11.15.468733v1.full) +important when [evaluating new and/or fashionable techniques](https://www.biorxiv.org/content/10.1101/2021.11.15.468733v1.full). _Question:_ Can you think of a way to include some negative controls in this experiment? We know that there are empty droplets in many methods, and we know @@ -999,11 +997,10 @@ Specifically, suppose you coded up the human and mouse cells (or the cell types you actually cared about) with batch (HTO, hash-tag oligo) labels, like in the 'S' column of the matrix above. (Think of it as a great big tibble, and think of the pieces of the big matrix as alternative assays.) Further, the protein -tags would help quite a bit if you think there really are expressed differences -within the usual cell subtypes (perhaps the ones you labeled with the hashtags). +tags could help quite a bit if you think there really are expressed differences +within the usual cell subtypes. -_Question_: Now can you think of a way to devise positive and negative controls -that doesn't depend upon consensus or what anyone else thinks? +_Question_: Can you use this to devise positive and negative controls in a way that makes biological sense? Congratulations, now you're even a little beyond the state of the published art. Apologies if Scott or I gave you the impression that [most published research is From ddea93201d1d44cc25c4712e70425f37f8b8b245 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 16:16:02 -0500 Subject: [PATCH 19/25] Reorganize this tome a little bit for clarity --- .../project2_chunks/figure/competitive-1.png | Bin 13147 -> 13136 bytes .../figure/confidenceintervals-1.png | Bin 15832 -> 15342 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 32191 -> 31213 bytes .../figure/rowDataAndColumnData-1.png | Bin 9857 -> 10539 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 26006 -> 27600 bytes vignettes/project2_chunks/project2_tim.Rmd | 46 +++++++------- vignettes/project2_chunks/project2_tim.md | 56 +++++++++--------- 7 files changed, 51 insertions(+), 51 deletions(-) diff --git a/vignettes/project2_chunks/figure/competitive-1.png b/vignettes/project2_chunks/figure/competitive-1.png index 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(No, seriously, you'll find out why eventually.) The package is, not coincidentally, called [tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html):

- Click for details + Make sure necessary packages are installed ```{r, tidysinglecell} if (!require("SingleCellExperiment")) { @@ -60,18 +60,16 @@ if (!require("tidyverse")) { ```
-# Tim's stab at classification and plotting - -It will be difficult to evaluate our strategy if we don't first load the data: -
- Click for details + Load necessary packages ```{r, loadpackages} library(SingleCellExperiment) library(tidySingleCellExperiment) ```
+It will be challenging to draw conclusions without first loading the data. + ```{r, gettidysce } # should package this... could just instantiate from a package via data(...) if (!exists("tidybarnyard")) { @@ -79,6 +77,25 @@ if (!exists("tidybarnyard")) { } ``` +The _[tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html)_ package gives +us some tools to stay in the Tidyverse as much as possible while working with +single-cell data. (It turns out that the underlying data makes this somewhat +less trivial than you might expect. Fortunately, you don't have to care.) + +There is a [little annoyance in _tidySingleCellExperiment_](https://github.com/stemangiola/tidySingleCellExperiment/issues/38) that we need to side-step to move along. Just to be sure, let's fix this and make sure we don't hit it later on: + +```{r, tidyfix} + +# if any column in `tidybarnyard` column data is named `cell`, rename it +# this is currently a bug in tidySingleCellExperiment: +# https://github.com/stemangiola/tidySingleCellExperiment/issues/38 +# +names(colData(tidybarnyard)) <- sub("cell", # pattern + "barcode", # replacement + names(colData(tidybarnyard))) # strings +``` +# Tim's stab at classification and plotting + It will be useful to know that, when you use the `$` operator on a typical Bioconductor object (such as a _tidySingleCellExperiment_ like ours), it assumes you want to see the column data (`colData`) with that name (see below). For @@ -101,23 +118,6 @@ feature-specific details lined up with rows, tends to stay in style. The only annoying feature (ha!) with this setup is that you need to explicitly tell R when you want data about the rows themselves (i.e., the rowData). -The _[tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html)_ package gives -us some tools to stay in the Tidyverse as much as possible while working with -single-cell data. (It turns out that the underlying data makes this somewhat -less trivial than you might expect. Fortunately, you don't have to care.) - -There is a [little annoyance in _tidySingleCellExperiment_](https://github.com/stemangiola/tidySingleCellExperiment/issues/38) that we need to side-step to move along. Just to be sure, let's fix this and make sure we don't hit it later on: - -```{r, tidyfix} - -# if any column in `tidybarnyard` column data is named `cell`, rename it -# this is currently a bug in tidySingleCellExperiment: -# https://github.com/stemangiola/tidySingleCellExperiment/issues/38 -# -names(colData(tidybarnyard)) <- sub("cell", # pattern - "barcode", # replacement - names(colData(tidybarnyard))) # strings -``` Now, let's see how these moving parts in the figure fit together, from the ground up. We will make use of the `dim` function, which provides dimensions diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 85fa849..6db71f7 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -36,7 +36,7 @@ data structures kind of suck. (No, seriously, you'll find out why eventually.) The package is, not coincidentally, called [tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html):
- Click for details + Make sure necessary packages are installed ```r @@ -55,12 +55,8 @@ if (!require("tidyverse")) { ```
-# Tim's stab at classification and plotting - -It will be difficult to evaluate our strategy if we don't first load the data: -
- Click for details + Load necessary packages ```r library(SingleCellExperiment) @@ -68,6 +64,8 @@ library(tidySingleCellExperiment) ```
+It will be challenging to draw conclusions without first loading the data. + ```r # should package this... could just instantiate from a package via data(...) @@ -76,6 +74,26 @@ if (!exists("tidybarnyard")) { } ``` +The _[tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html)_ package gives +us some tools to stay in the Tidyverse as much as possible while working with +single-cell data. (It turns out that the underlying data makes this somewhat +less trivial than you might expect. Fortunately, you don't have to care.) + +There is a [little annoyance in _tidySingleCellExperiment_](https://github.com/stemangiola/tidySingleCellExperiment/issues/38) that we need to side-step to move along. Just to be sure, let's fix this and make sure we don't hit it later on: + + +```r + +# if any column in `tidybarnyard` column data is named `cell`, rename it +# this is currently a bug in tidySingleCellExperiment: +# https://github.com/stemangiola/tidySingleCellExperiment/issues/38 +# +names(colData(tidybarnyard)) <- sub("cell", # pattern + "barcode", # replacement + names(colData(tidybarnyard))) # strings +``` +# Tim's stab at classification and plotting + It will be useful to know that, when you use the `$` operator on a typical Bioconductor object (such as a _tidySingleCellExperiment_ like ours), it assumes you want to see the column data (`colData`) with that name (see below). For @@ -98,24 +116,6 @@ feature-specific details lined up with rows, tends to stay in style. The only annoying feature (ha!) with this setup is that you need to explicitly tell R when you want data about the rows themselves (i.e., the rowData). -The _[tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html)_ package gives -us some tools to stay in the Tidyverse as much as possible while working with -single-cell data. (It turns out that the underlying data makes this somewhat -less trivial than you might expect. Fortunately, you don't have to care.) - -There is a [little annoyance in _tidySingleCellExperiment_](https://github.com/stemangiola/tidySingleCellExperiment/issues/38) that we need to side-step to move along. Just to be sure, let's fix this and make sure we don't hit it later on: - - -```r - -# if any column in `tidybarnyard` column data is named `cell`, rename it -# this is currently a bug in tidySingleCellExperiment: -# https://github.com/stemangiola/tidySingleCellExperiment/issues/38 -# -names(colData(tidybarnyard)) <- sub("cell", # pattern - "barcode", # replacement - names(colData(tidybarnyard))) # strings -``` Now, let's see how these moving parts in the figure fit together, from the ground up. We will make use of the `dim` function, which provides dimensions @@ -182,7 +182,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture2.inDrops.CTGAGCGT-AGAATGCG-CCTACTAG" +#> [1] "Mixture2.inDrops.GTGCCCAT-AGAATGCG-CGTATCGG" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -192,7 +192,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000045268_mm10_Zfp691" +#> [1] "mm10_ENSMUSG00000107705_mm10_Umad1" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] @@ -222,7 +222,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9225 +#> [1] 0.9112 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -585,7 +585,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 1941 2116 142 +#> 1941 142 2116 barnyardtibble$mclass <- factor(mfit$classification) # plot the results From 8dc31a271027f3fb3b2ddded1c4292413703b41c Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 16:20:55 -0500 Subject: [PATCH 20/25] Clarify tidySCE section --- .../project2_chunks/figure/competitive-1.png | Bin 13136 -> 13138 bytes .../figure/confidenceintervals-1.png | Bin 15342 -> 15772 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31213 -> 31889 bytes .../figure/rowDataAndColumnData-1.png | Bin 10539 -> 9882 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 27600 -> 29052 bytes vignettes/project2_chunks/project2_tim.Rmd | 3 ++- 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For that, we need cutoffs. With the release of Bioconductor 3.14, the project includes a tidy single cell experiment (data structure) package, which is great since all other single cell data structures kind of suck. (No, seriously, you'll find out why eventually.) -The package is, not coincidentally, called [tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html): +The package is, not coincidentally, called [tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html). The linked instructions are helpful; read them when you +have a chance. Meanwhile, let's make sure we can load it in this analysis.

Make sure necessary packages are installed diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 6db71f7..b25aef7 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -33,7 +33,8 @@ i.e., the ability to classify a cell confidently. For that, we need cutoffs. With the release of Bioconductor 3.14, the project includes a tidy single cell experiment (data structure) package, which is great since all other single cell data structures kind of suck. (No, seriously, you'll find out why eventually.) -The package is, not coincidentally, called [tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html): +The package is, not coincidentally, called [tidySingleCellExperiment](http://www.bioconductor.org/packages/release/bioc/vignettes/tidySingleCellExperiment/inst/doc/introduction.html). The linked instructions are helpful; read them when you +have a chance. Meanwhile, let's make sure we can load it in this analysis.
Make sure necessary packages are installed @@ -182,7 +183,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture2.inDrops.GTGCCCAT-AGAATGCG-CGTATCGG" +#> [1] "Mixture2.inDrops.CAAGGAAT-AAGAGCGT-TAAGACGG" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -192,7 +193,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000107705_mm10_Umad1" +#> [1] "mm10_ENSMUSG00000000881_mm10_Dlg3" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] @@ -222,7 +223,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9112 +#> [1] 0.932 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -585,7 +586,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 1941 142 2116 +#> 142 2116 1941 barnyardtibble$mclass <- factor(mfit$classification) # plot the results From d1593061423a4726d77d6c41dc4139fc2aa4fde5 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 16:39:36 -0500 Subject: [PATCH 21/25] tidy up a bit more, choose words more carefully --- .../project2_chunks/figure/competitive-1.png | Bin 13138 -> 13138 bytes .../figure/confidenceintervals-1.png | Bin 15772 -> 15335 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31889 -> 31213 bytes .../figure/rowDataAndColumnData-1.png | Bin 9882 -> 10280 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 29052 -> 25940 bytes vignettes/project2_chunks/project2_tim.R | 4 ++-- 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100644 --- a/vignettes/project2_chunks/project2_tim.R +++ b/vignettes/project2_chunks/project2_tim.R @@ -61,7 +61,7 @@ identical(dim(tidybarnyard), identical(dim(tidybarnyard), dim(assay(tidybarnyard))) # there is a shortcut for the `counts` assay, since it's so common: -counts(tidybarnyard)[1:3, 1:3] +counts(tidybarnyard)[1:3, 1:3] # this holds the UMI counts, or a `.` for 0. # does each row (gene) in the object have a corresponding rowData() row? identical(rownames(tidybarnyard), rownames(rowData(tidybarnyard))) @@ -92,7 +92,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" show(aGene) # how many copies of this random gene were found in this random cell? -counts(tidybarnyard)[aGene, aCell] +counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. # note that the odds are good that you'll get a 0 for this random combination: library(Matrix) # for the `nnzero` function diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index fb74bb1..a2bc020 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -31,7 +31,7 @@ you will recall, is tested for significance using a marginal t-test), we can rely upon a generalized version of residual variance called [deviance]("https://en.wikipedia.org/wiki/Deviance_(statistics) "Wikipedia page for deviance") to let us answer ANOVA-style questions: "how much variability is explained by a particular factor in the model?" First we need to set up our response variable, -i.e., the ability to classify a cell confidently. For that, we need cutoffs. +i.e., the ability to classify a cell confidently. For that, we need labels. # tidySingleCellExperiment @@ -124,6 +124,10 @@ Now, let's see how these moving parts in the figure fit together, from the ground up. We will make use of the `dim` function, which provides dimensions for a rectangular object in the form rows x columns. This will also make it a little easier to keep track of what's going on when we subset either one. +To avoid the document becoming absurdly large, we'll fold it up; + +
+ click to open it back up. ```{r, rowDataAndColumnData } @@ -141,7 +145,7 @@ identical(dim(tidybarnyard), identical(dim(tidybarnyard), dim(assay(tidybarnyard))) # there is a shortcut for the `counts` assay, since it's so common: -counts(tidybarnyard)[1:3, 1:3] +counts(tidybarnyard)[1:3, 1:3] # this holds the UMI counts, or a `.` for 0. # does each row (gene) in the object have a corresponding rowData() row? identical(rownames(tidybarnyard), rownames(rowData(tidybarnyard))) @@ -172,7 +176,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" show(aGene) # how many copies of this random gene were found in this random cell? -counts(tidybarnyard)[aGene, aCell] +counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. # note that the odds are good that you'll get a 0 for this random combination: library(Matrix) # for the `nnzero` function @@ -224,6 +228,7 @@ ggplot(tibble(estimate=estimates), aes(estimate)) + # the Central Limit Theorem lives to fight another day, # and we have a decent idea of how to navigate our data. ``` +
Often, when someone cracks open a single cell dataset, the first thing they do is to cluster the cells (based on whatever approach is fashionable). We don't diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index b25aef7..1c308c0 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -26,7 +26,7 @@ you will recall, is tested for significance using a marginal t-test), we can rely upon a generalized version of residual variance called [deviance]("https://en.wikipedia.org/wiki/Deviance_(statistics) "Wikipedia page for deviance") to let us answer ANOVA-style questions: "how much variability is explained by a particular factor in the model?" First we need to set up our response variable, -i.e., the ability to classify a cell confidently. For that, we need cutoffs. +i.e., the ability to classify a cell confidently. For that, we need labels. # tidySingleCellExperiment @@ -122,6 +122,10 @@ Now, let's see how these moving parts in the figure fit together, from the ground up. We will make use of the `dim` function, which provides dimensions for a rectangular object in the form rows x columns. This will also make it a little easier to keep track of what's going on when we subset either one. +To avoid the document becoming absurdly large, we'll fold it up; + +
+ click to open it back up. ```r @@ -147,7 +151,7 @@ identical(dim(tidybarnyard), dim(assay(tidybarnyard))) #> [1] TRUE # there is a shortcut for the `counts` assay, since it's so common: -counts(tidybarnyard)[1:3, 1:3] +counts(tidybarnyard)[1:3, 1:3] # this holds the UMI counts, or a `.` for 0. #> 3 x 3 sparse Matrix of class "dgCMatrix" #> Mixture1.10x-Chromium-v2.GGGCATCGTCACACGC #> hg19_ENSG00000000003_hg19_TSPAN6 1 @@ -183,7 +187,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture2.inDrops.CAAGGAAT-AAGAGCGT-TAAGACGG" +#> [1] "Mixture2.inDrops.TAATGTGG-AAGAGCGT-CGGGTAGT" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -193,10 +197,10 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000000881_mm10_Dlg3" +#> [1] "mm10_ENSMUSG00000027349_mm10_Fam98b" # how many copies of this random gene were found in this random cell? -counts(tidybarnyard)[aGene, aCell] +counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. #> [1] 0 # note that the odds are good that you'll get a 0 for this random combination: @@ -223,7 +227,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.932 +#> [1] 0.9255 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -257,6 +261,7 @@ ggplot(tibble(estimate=estimates), aes(estimate)) + # the Central Limit Theorem lives to fight another day, # and we have a decent idea of how to navigate our data. ``` +
Often, when someone cracks open a single cell dataset, the first thing they do is to cluster the cells (based on whatever approach is fashionable). We don't @@ -586,7 +591,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 142 2116 1941 +#> 1941 142 2116 barnyardtibble$mclass <- factor(mfit$classification) # plot the results From 587935305cdc3ce89a7c606f264dbf18b8dad889 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 16:44:56 -0500 Subject: [PATCH 22/25] rerun vignette from a cold start --- .../project2_chunks/figure/competitive-1.png | Bin 13138 -> 13145 bytes .../figure/confidenceintervals-1.png | Bin 15335 -> 15768 bytes .../figure/rowDataAndColumnData-1.png | Bin 10280 -> 10187 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 25940 -> 27452 bytes vignettes/project2_chunks/project2_tim.md | 142 +++++++++++++++++- 5 files changed, 136 insertions(+), 6 deletions(-) diff --git a/vignettes/project2_chunks/figure/competitive-1.png b/vignettes/project2_chunks/figure/competitive-1.png index 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To cite Bioconductor, see +#> 'citation("Biobase")', and for packages 'citation("pkgname")'. +#> +#> Attaching package: 'Biobase' +#> The following object is masked from 'package:MatrixGenerics': +#> +#> rowMedians +#> The following objects are masked from 'package:matrixStats': +#> +#> anyMissing, rowMedians if (!require("tidySingleCellExperiment")) { BiocManager::install("tidySingleCellExperiment") library(tidySingleCellExperiment) } +#> Loading required package: tidySingleCellExperiment +#> +#> Attaching package: 'tidySingleCellExperiment' +#> The following object is masked from 'package:IRanges': +#> +#> slice +#> The following object is masked from 'package:S4Vectors': +#> +#> rename +#> The following object is masked from 'package:matrixStats': +#> +#> count +#> The following object is masked from 'package:stats': +#> +#> filter if (!require("tidyverse")) { BiocManager::install("tidyverse") library(tidyverse) } +#> Loading required package: tidyverse +#> ── Attaching packages ─────────────────────────────────────── tidyverse 1.3.1 ── +#> ✔ ggplot2 3.3.5 ✔ purrr 0.3.4 +#> ✔ tibble 3.1.6 ✔ dplyr 1.0.7 +#> ✔ tidyr 1.1.4 ✔ stringr 1.4.0 +#> ✔ readr 2.1.0 ✔ forcats 0.5.1 +#> ── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ── +#> ✖ dplyr::add_count() masks tidySingleCellExperiment::add_count() +#> ✖ dplyr::bind_cols() masks tidySingleCellExperiment::bind_cols() +#> ✖ dplyr::bind_rows() masks tidySingleCellExperiment::bind_rows() +#> ✖ dplyr::collapse() masks IRanges::collapse() +#> ✖ dplyr::combine() masks Biobase::combine(), BiocGenerics::combine() +#> ✖ dplyr::count() masks tidySingleCellExperiment::count(), matrixStats::count() +#> ✖ dplyr::desc() masks IRanges::desc() +#> ✖ tidyr::expand() masks S4Vectors::expand() +#> ✖ dplyr::filter() masks tidySingleCellExperiment::filter(), stats::filter() +#> ✖ dplyr::first() masks S4Vectors::first() +#> ✖ dplyr::lag() masks stats::lag() +#> ✖ ggplot2::Position() masks BiocGenerics::Position(), base::Position() +#> ✖ purrr::reduce() masks GenomicRanges::reduce(), IRanges::reduce() +#> ✖ dplyr::rename() masks tidySingleCellExperiment::rename(), S4Vectors::rename() +#> ✖ dplyr::slice() masks tidySingleCellExperiment::slice(), IRanges::slice() ```

@@ -132,10 +230,10 @@ To avoid the document becoming absurdly large, we'll fold it up; # as what is our object masquerading? show(tidybarnyard[,0]) # "just show me information about it, with 0 cells" -#> # A SingleCellExperiment-tibble abstraction: 0 × 7 +#> # A SingleCellExperiment-tibble abstraction: 0 × 5 #> # Features=62046 | Assays=counts -#> # … with 7 variables: cell , name , experiment , method , -#> # barcode , fracmouse , frachuman +#> # … with 5 variables: cell , name , experiment , method , +#> # barcode # how many rows (genes) and columns (cells) are there in our tidy barnyard? dim(tidybarnyard) @@ -187,7 +285,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture2.inDrops.TAATGTGG-AAGAGCGT-CGGGTAGT" +#> [1] "Mixture2.inDrops.CCAGACAG-AAGAGCGT-CATCGCAG" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -197,7 +295,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000027349_mm10_Fam98b" +#> [1] "mm10_ENSMUSG00000058318_mm10_Phf21a" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. @@ -205,6 +303,14 @@ counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. # note that the odds are good that you'll get a 0 for this random combination: library(Matrix) # for the `nnzero` function +#> +#> Attaching package: 'Matrix' +#> The following objects are masked from 'package:tidyr': +#> +#> expand, pack, unpack +#> The following object is masked from 'package:S4Vectors': +#> +#> expand sparsity <- 1 - (nnzero(counts(tidybarnyard)) / # number of nonzero counts prod(dim(tidybarnyard))) # number of rows * columns @@ -227,7 +333,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9255 +#> [1] 0.9099 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -492,6 +598,19 @@ library(coefplot) # classifiable ~ method coefplot(fit1, trans=invlogit) + theme_minimal() +#> Warning: `funs()` was deprecated in dplyr 0.8.0. +#> Please use a list of either functions or lambdas: +#> +#> # Simple named list: +#> list(mean = mean, median = median) +#> +#> # Auto named with `tibble::lst()`: +#> tibble::lst(mean, median) +#> +#> # Using lambdas +#> list(~ mean(., trim = .2), ~ median(., na.rm = TRUE)) +#> This warning is displayed once every 8 hours. +#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated. #> Warning: It is deprecated to specify `guide = FALSE` to remove a guide. Please #> use `guide = "none"` instead. @@ -571,6 +690,17 @@ automatic gating of actual flow cytometry data this way, in fact, and it works # fits a Gaussian mixture model with arbitrary covariance structure and uses # a Bayesian penalization scheme to choose how many components exist in the mix library(mclust) +#> __ ___________ __ _____________ +#> / |/ / ____/ / / / / / ___/_ __/ +#> / /|_/ / / / / / / / /\__ \ / / +#> / / / / /___/ /___/ /_/ /___/ // / +#> /_/ /_/\____/_____/\____//____//_/ version 5.4.8 +#> Type 'citation("mclust")' for citing this R package in publications. +#> +#> Attaching package: 'mclust' +#> The following object is masked from 'package:purrr': +#> +#> map ```
From 2e1cc0fdfc3b0453f590785d2b8ca093e7094b5b Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 18:46:59 -0500 Subject: [PATCH 23/25] Correct a statement about 100x100 samples (it's 200) --- .../project2_chunks/figure/competitive-1.png | Bin 13145 -> 13087 bytes .../figure/confidenceintervals-1.png | Bin 15768 -> 15544 bytes .../figure/mixlabeledplot-1.png | Bin 32988 -> 33239 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31213 -> 31865 bytes vignettes/project2_chunks/figure/remix-1.png | Bin 16374 -> 16599 bytes vignettes/project2_chunks/figure/remix-2.png | Bin 18079 -> 17843 bytes vignettes/project2_chunks/figure/remix-3.png | Bin 29440 -> 29657 bytes .../figure/rowDataAndColumnData-1.png | Bin 10187 -> 9580 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 27452 -> 22018 bytes vignettes/project2_chunks/project2_tim.R | 5 ++-- vignettes/project2_chunks/project2_tim.Rmd | 5 ++-- vignettes/project2_chunks/project2_tim.md | 25 +++++++++--------- 12 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z{akxnC}ZXx&-?ouZry@5tWTpU(2>GSrhN$-><`t^yDr|Z{`l@OvNJFoGz6DQ*{-ev zm(|dpqn|b5NZ$M9Gl4d#qN+OYXul~$ceH18dL$rSRy;Fn>my@M+ji_ggd0=cl(e)z zD-IU0g-CCwvW4j>DGp9f-@I_Di% -# mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% + mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% ggplot(aes(method, success, color=method)) + scale_y_continuous(labels = scales::label_percent()) + ylab("classification performance") + diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index e115aaa..4393a64 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -688,7 +688,7 @@ library(ggforce) # for better resolution, you could resample batches of 500, 1000, ... results %>% -# mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% + mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% ggplot(aes(method, success, color=method)) + scale_y_continuous(labels = scales::label_percent()) + ylab("classification performance") + @@ -699,10 +699,10 @@ results %>% ``` -_Question:_ I commented out a line in the plot above. Would it make it clearer? +_Question:_ What does `fct_reorder` (from the `forcats` package) do above? You might conclude that we could directly compute confidence intervals from the -above results. You would be correct. Let's do this in a tidy-ish fashion: +above results. You would be correct. Let's do this tidily: ```{r, confidenceintervals} diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 5529346..5af57dc 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -285,7 +285,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture2.inDrops.GTTCTGCT-AAGAGCGT-TAACCATC" +#> [1] "Mixture1.inDrops.CAGATGGG-TATCCTCT-AGTTTAGA" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -295,7 +295,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000030061_mm10_Uba3" +#> [1] "mm10_ENSMUSG00000095706_mm10_Olfr209" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. @@ -333,7 +333,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9239 +#> [1] 0.9301 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -721,7 +721,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 1114 1106 1979 +#> 1941 2116 142 barnyardtibble$mclass <- factor(mfit$classification) # plot the results @@ -761,17 +761,17 @@ barnyardtibble %>% with(barnyardtibble, table(mixlabel, label)) #> label #> mixlabel human mouse suspect -#> human 1076 0 30 -#> mouse 0 1721 258 -#> suspect 867 0 247 +#> human 1925 0 191 +#> mouse 0 1716 225 +#> suspect 18 5 119 # specifically, do we label all the human and mouse cells confidently? with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] #> label #> mixlabel human mouse -#> human 1076 0 -#> mouse 0 1721 -#> suspect 867 0 +#> human 1925 0 +#> mouse 0 1716 +#> suspect 18 5 ``` How did we do? @@ -967,7 +967,7 @@ library(ggforce) # for better resolution, you could resample batches of 500, 1000, ... results %>% -# mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% + mutate(method = fct_reorder(method, desc(success), .fun='median')) %>% ggplot(aes(method, success, color=method)) + scale_y_continuous(labels = scales::label_percent()) + ylab("classification performance") + @@ -979,10 +979,10 @@ results %>% ![plot of chunk sinaplot](figure/sinaplot-1.png) -_Question:_ I commented out a line in the plot above. Would it make it clearer? +_Question:_ What does `fct_reorder` (from the `forcats` package) do above? You might conclude that we could directly compute confidence intervals from the -above results. You would be correct. Let's do this in a tidy-ish fashion: +above results. You would be correct. Let's do this tidily: ```r From 424834bc5ac8fa77cc3b5e95837b774c5c145353 Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 18:58:56 -0500 Subject: [PATCH 25/25] Once more, with feeling --- .../project2_chunks/figure/competitive-1.png | Bin 13148 -> 13135 bytes .../figure/confidenceintervals-1.png | Bin 15552 -> 15955 bytes .../figure/mixlabeledplot-1.png | Bin 32988 -> 33103 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 32191 -> 31267 bytes .../figure/ordered_sinaplot-1.png | Bin 0 -> 25555 bytes vignettes/project2_chunks/figure/remix-1.png | Bin 16374 -> 16270 bytes vignettes/project2_chunks/figure/remix-2.png | Bin 18079 -> 17858 bytes vignettes/project2_chunks/figure/remix-3.png | Bin 29440 -> 29511 bytes .../figure/rowDataAndColumnData-1.png | Bin 9559 -> 9432 bytes vignettes/project2_chunks/project2_tim.R | 2 +- vignettes/project2_chunks/project2_tim.Rmd | 13 ++++--- vignettes/project2_chunks/project2_tim.md | 33 ++++++++++-------- 12 files 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sinaplot---------------------------------------------------------------- +## ---- ordered_sinaplot-------------------------------------------------------- # fire up geom_sina because it rules: library(ggforce) diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index 4393a64..d6723f8 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -681,7 +681,7 @@ results <- bind_rows(runs) Per usual, it helps to plot the results. -```{r, sinaplot} +```{r, ordered_sinaplot} # fire up geom_sina because it rules: library(ggforce) @@ -737,8 +737,13 @@ CIs %>% _Question (not required)_: Can you overlay the two summaries above? (I can't.) -_Question (not required)_: Is this also feasible for each combination of -(mixture, method)? If so, how would you structure your resampling scheme? +_Question (not required)_: Is it feasible to block on experiment x method? + +_Question (not required)_: How could you structure a scheme for the above? + +(Note that I might ask you to do something very much like this in project 3...) + +# An equivalence between hypothesis tests and confidence intervals Perhaps you've previously been told that it is not possible to exclude the possibility that two groups (or groups of samples) are equivalent at the 5% @@ -747,7 +752,7 @@ since inverting a test at a given alpha is equivalent to computing a (1-alpha)% confidence interval. Congratulations, you just computed all marginal comparisons for these methods at a significance level of 0.05. -_Question:_ What happens if you take bigger samples, or more of them? +_Question:_ What happens if you take bigger samples, or more of them, or both? Suppose we went back to the original dataset (or, for the sake of argument, a larger dataset) and resampled the hell out of it. At some point, do you suppose diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index 5af57dc..7cc28c6 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -285,7 +285,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture1.inDrops.CAGATGGG-TATCCTCT-AGTTTAGA" +#> [1] "Mixture1.inDrops.ATTGAAGT-CTCTCTAT-CACAACAG" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -295,7 +295,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000095706_mm10_Olfr209" +#> [1] "mm10_ENSMUSG00000090191_mm10_9230105E05Rik" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. @@ -333,7 +333,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9301 +#> [1] 0.9302 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -721,7 +721,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 1941 2116 142 +#> 2156 113 1930 barnyardtibble$mclass <- factor(mfit$classification) # plot the results @@ -761,17 +761,17 @@ barnyardtibble %>% with(barnyardtibble, table(mixlabel, label)) #> label #> mixlabel human mouse suspect -#> human 1925 0 191 -#> mouse 0 1716 225 -#> suspect 18 5 119 +#> human 1932 0 224 +#> mouse 0 1712 218 +#> suspect 11 9 93 # specifically, do we label all the human and mouse cells confidently? with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] #> label #> mixlabel human mouse -#> human 1925 0 -#> mouse 0 1716 -#> suspect 18 5 +#> human 1932 0 +#> mouse 0 1712 +#> suspect 11 9 ``` How did we do? @@ -977,7 +977,7 @@ results %>% ggtitle("Cell classification results by prep, 200 cells apiece") ``` -![plot of chunk sinaplot](figure/sinaplot-1.png) +![plot of chunk ordered_sinaplot](figure/ordered_sinaplot-1.png) _Question:_ What does `fct_reorder` (from the `forcats` package) do above? @@ -1019,8 +1019,13 @@ CIs %>% _Question (not required)_: Can you overlay the two summaries above? (I can't.) -_Question (not required)_: Is this also feasible for each combination of -(mixture, method)? If so, how would you structure your resampling scheme? +_Question (not required)_: Is it feasible to block on experiment x method? + +_Question (not required)_: How could you structure a scheme for the above? + +(Note that I might ask you to do something very much like this in project 3...) + +# An equivalence between hypothesis tests and confidence intervals Perhaps you've previously been told that it is not possible to exclude the possibility that two groups (or groups of samples) are equivalent at the 5% @@ -1029,7 +1034,7 @@ since inverting a test at a given alpha is equivalent to computing a (1-alpha)% confidence interval. Congratulations, you just computed all marginal comparisons for these methods at a significance level of 0.05. -_Question:_ What happens if you take bigger samples, or more of them? +_Question:_ What happens if you take bigger samples, or more of them, or both? Suppose we went back to the original dataset (or, for the sake of argument, a larger dataset) and resampled the hell out of it. At some point, do you suppose

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z0sZAq5U@&0p6}l3iX_VMTu8=gzEdiM_>m@d1~x?jY9iN0?EVhgCC`116!sy;9UmLh zOgMqPg9N}X0V#ts#w8bp>Hvk;{*>R4%g1*oBb6gHmS^5NqOIy5qKU4+oMI`s@CG|t zujgfD&CJW=fCuE^et{Ya_Qps8lA3Wxf*MM_5m1a+-!80k$m5_zRtr9|(2RHgUL&9n zA08czudgS>#+ExQ^}X!R$*F}xM~`ufPp z$zd0OCx^WohQlvrex81^++orQkP?7$X_PZoclpmhjlikFSYIR;!`8Eyqg$ZZ+mKma z9&4;6m}`D{lj7e(Cbm2Wk4kKH^uN8}YtQh_pFiQa6XH|=+hHMOBo!r!#Gm{A7aE7Y A`~Uy| diff --git a/vignettes/project2_chunks/project2_tim.R b/vignettes/project2_chunks/project2_tim.R index 6b0d193..2c8f245 100644 --- a/vignettes/project2_chunks/project2_tim.R +++ b/vignettes/project2_chunks/project2_tim.R @@ -490,10 +490,11 @@ names(subsamples) <- paste0("run", seq_along(subsamples)) # purrr::map maps a function over a list library(purrr) -# evaluate performance across 100 bootstrap samples of 100 cells per method +# evaluate performance with 200 bootstrap samples of (ideally) 200 cells/method +# (i.e. subsamples <- replicate(n=200, sample_umis(..., ideal=200, ...)), above) runs <- purrr::map(subsamples, performance_by_method, atibble=barnyardtibble) -# for plotting, stack them +# for plotting, stack them row-by-row results <- bind_rows(runs) diff --git a/vignettes/project2_chunks/project2_tim.Rmd b/vignettes/project2_chunks/project2_tim.Rmd index a2bc020..e115aaa 100644 --- a/vignettes/project2_chunks/project2_tim.Rmd +++ b/vignettes/project2_chunks/project2_tim.Rmd @@ -670,10 +670,11 @@ names(subsamples) <- paste0("run", seq_along(subsamples)) # purrr::map maps a function over a list library(purrr) -# evaluate performance across 100 bootstrap samples of 100 cells per method +# evaluate performance with 200 bootstrap samples of (ideally) 200 cells/method +# (i.e. subsamples <- replicate(n=200, sample_umis(..., ideal=200, ...)), above) runs <- purrr::map(subsamples, performance_by_method, atibble=barnyardtibble) -# for plotting, stack them +# for plotting, stack them row-by-row results <- bind_rows(runs) ``` diff --git a/vignettes/project2_chunks/project2_tim.md b/vignettes/project2_chunks/project2_tim.md index fc3d770..5529346 100644 --- a/vignettes/project2_chunks/project2_tim.md +++ b/vignettes/project2_chunks/project2_tim.md @@ -285,7 +285,7 @@ as_tibble(tidybarnyard) %>% # "turn the colData into a tibble" # your cell: show(aCell) -#> [1] "Mixture2.inDrops.CCAGACAG-AAGAGCGT-CATCGCAG" +#> [1] "Mixture2.inDrops.GTTCTGCT-AAGAGCGT-TAACCATC" # does that mean we can ask for a random gene with certain attributes? as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" @@ -295,7 +295,7 @@ as_tibble(rowData(tidybarnyard)) %>% # "turn the rowData into a tibble" # your gene: show(aGene) -#> [1] "mm10_ENSMUSG00000058318_mm10_Phf21a" +#> [1] "mm10_ENSMUSG00000030061_mm10_Uba3" # how many copies of this random gene were found in this random cell? counts(tidybarnyard)[aGene, aCell] # UMI counts for a given [gene, cell]. @@ -333,7 +333,7 @@ samples <- (length(aHundredCells) * length(aHundredGenes)) nonzero <- nnzero(counts(tidybarnyard)[aHundredGenes, aHundredCells]) sparsity_hat <- (samples - nonzero) / samples sparsity_hat # estimated sparsity -#> [1] 0.9099 +#> [1] 0.9239 # In fact, we can use this scheme to look at sampling error: sample_sparsity <- function(object, cells=100, genes=100) { @@ -721,7 +721,7 @@ mfit <- Mclust(logit(barnyardtibble[, c("fracmouse","frachuman")]), table(mfit$classification) # it turns out that we end up with less human cells #> #> 1 2 3 -#> 1941 142 2116 +#> 1114 1106 1979 barnyardtibble$mclass <- factor(mfit$classification) # plot the results @@ -761,17 +761,17 @@ barnyardtibble %>% with(barnyardtibble, table(mixlabel, label)) #> label #> mixlabel human mouse suspect -#> human 1925 0 191 -#> mouse 0 1716 225 -#> suspect 18 5 119 +#> human 1076 0 30 +#> mouse 0 1721 258 +#> suspect 867 0 247 # specifically, do we label all the human and mouse cells confidently? with(barnyardtibble, table(mixlabel, label))[, c("human", "mouse")] #> label #> mixlabel human mouse -#> human 1925 0 -#> mouse 0 1716 -#> suspect 18 5 +#> human 1076 0 +#> mouse 0 1721 +#> suspect 867 0 ``` How did we do? @@ -949,10 +949,11 @@ names(subsamples) <- paste0("run", seq_along(subsamples)) # purrr::map maps a function over a list library(purrr) -# evaluate performance across 100 bootstrap samples of 100 cells per method +# evaluate performance with 200 bootstrap samples of (ideally) 200 cells/method +# (i.e. subsamples <- replicate(n=200, sample_umis(..., ideal=200, ...)), above) runs <- purrr::map(subsamples, performance_by_method, atibble=barnyardtibble) -# for plotting, stack them +# for plotting, stack them row-by-row results <- bind_rows(runs) ``` From 3c602a11bfb3c28171bb775e811e33e9a3d733de Mon Sep 17 00:00:00 2001 From: "Tim Triche, Jr" Date: Sat, 20 Nov 2021 18:50:41 -0500 Subject: [PATCH 24/25] Tidy up a few more bits for clarity --- .../project2_chunks/figure/competitive-1.png | Bin 13087 -> 13148 bytes .../figure/confidenceintervals-1.png | Bin 15544 -> 15552 bytes .../figure/mixlabeledplot-1.png | Bin 33239 -> 32988 bytes .../project2_chunks/figure/mixtureModel-1.png | Bin 31865 -> 32191 bytes vignettes/project2_chunks/figure/remix-1.png | Bin 16599 -> 16374 bytes vignettes/project2_chunks/figure/remix-2.png | Bin 17843 -> 18079 bytes vignettes/project2_chunks/figure/remix-3.png | Bin 29657 -> 29440 bytes .../figure/rowDataAndColumnData-1.png | Bin 9580 -> 9559 bytes .../project2_chunks/figure/sinaplot-1.png | Bin 22018 -> 25928 bytes vignettes/project2_chunks/project2_tim.R | 2 +- vignettes/project2_chunks/project2_tim.Rmd | 6 ++-- vignettes/project2_chunks/project2_tim.md | 26 +++++++++--------- 12 files changed, 17 insertions(+), 17 deletions(-) diff --git a/vignettes/project2_chunks/figure/competitive-1.png b/vignettes/project2_chunks/figure/competitive-1.png index 3aa5966e9cfb89ea34157791a13a952380089789..e211191ff5fba25b6691f00eaae8a6df894e1390 100644 GIT binary patch delta 9944 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