scRNA-seq 10X Single-Cell RNA Preprocessing Pipeline

Overview

This project performs pre-processing of 10X Genomics Single-Cell RNA-seq data using Galaxy platform tools including RNA STARsolo and DropletUtils.

Dataset

• Source: 1k PBMCs from a Healthy Donor (v3 chemistry) - 10X Genomics
• Input: Sub-sampled FASTQ files (~300 cells) from Zenodo
• Genome: Human hg19 (GRCh37)
• Chemistry: 10X Chromium v3

Workflow Steps

1. Data Upload (FASTQ + GTF + Barcode Whitelist)
2. Demultiplexing & Mapping with RNA STARsolo
3. Quality Control with MultiQC
4. Cell Filtering with DropletUtils (DefaultDrops)
5. Barcode Ranking (Knee/Inflection Plot)
6. Custom Cell Filtering with DropletUtils (EmptyDrops)

Key Results

Metric	Value
Uniquely Mapped Reads	18.3%
Cells Detected (STARsolo)	5200
Cells After DefaultDrops	254
Cells After EmptyDrops	250

Tools Used

Tool	Version	Purpose
RNA STARsolo	2.7.11a	Mapping & Quantification
MultiQC	1.27	Quality Control
DropletUtils	1.10.0	Cell Filtering

Repository Structure

├── README.md ├── workflow/ │ └── scRNA-seq-10X-Preprocessing-Workflow.ga ├── results/ │ ├── multiqc_report.html │ ├── barcode_rank_plot.png │ └── emptydrops_plot.png └── data/ └── barcodes_defaultdrops.tsv

Reference

• Galaxy Training Tutorial
• Zenodo Dataset: https://zenodo.org/record/3457880