AStruct

AStruct is a powerful tool for detecting allele-specific RNA secondary structures (RiboSNitches) within one sample from structomics sequencing data.

Introduction

Here, we present AStruct, a Java-based software for identifying the local structure difference of SNPs from RT-Stop and RT-Mut structure sequencing data. It will automatically target the comparison window according to the maximum spanning Ref and Alt reads length around SNPs from the inputted sorted BAM file(s). SNPs that do not have enough reads support will be filtered out. Replicate samples are highly recommended by removing many false-positive results, meaning the alleles difference must be larger then replicates perturbation. The output will provide you a AStruct score, evaluating the overall allele’s structure difference. The Astruct score and other basic SNP annotation are written in the first file suffix with riboSnotch.txt. More detailed information, including the intermediate values for calculating AStruct score, the sequence, the region position, base counts, Ref/Alt each base structure score, and the significant P Value of each base structure difference, are also listed in a second file suffix with riboSnotchDetail.txt. Note that the randomness of permutation might introduce a slightly difference between each run. So, set the seed in advance would be helpful :)

Table of Contents

• Requirements
• Download
• Quick Start Guide
• Usage
• Output Headers
• License

Requirements

• JDK 8

Download

• This tool can be download by instruction as follow:

wget https://github.com/canceromics/AStruct/releases/download/V1.1/AStruct.jar

The tool is named as AStruct.jar in this directory.

QuickStart

• Start from bam file of and input sample for example.

java -Xmx16g -jar AStruct.jar ribosnitch -tf example/data/T1.bam example/data/T2.bam -cf example/data/C1.bam example/data/C2.bam -o example/result/Example -gf example/reference/GRCh38.p12_NC_000022.11.fa -ef example/reference/GRCh38.p12_NC_000022.11.gff 
-mutf example/reference/SNP_NC_000022.11.vcf -repli

Running this instruction will result in getting a file named Example_riboSNitch.txt. example/result/Example means output_dir/file_prefix

More commonly used

java [-Xmx16g] -jar AStruct.jar <method> -tf <treat.bam(s)> -gf <genome.fa> -ef <gencode.gtf/gff> -mutf <snp.cvf> -o <path/out_prefix> [-cf control.bam(s)] [options]

Usage

• More details of this tool can be found with -h parameter

java [-Xmx16g] -jar AStruct.jar <method> [options]
Methods:
    ribosnitch
    find different structure around SNPs. (all bams need to be sorted by coordinate)
    options:
        <-tf treat.bam ...> treat bam files
        <-cf control.bam ...> control bam files
        <-ef gencode.gtf> annotation gtf/gff file
        <-gf genome.fa> genome fasta file
        <-mutf snp.vcf> snp vcf/bed file
        <-o out_prefix> output file prefix
        [-permu count] set permutation with count times. (default is 1000)
        [-repli] enable replication filter
        [-seed seed] set random seed
        [-bases bases] allow structures show in some bases such as "AC". (default is all bases)
        [-mut] switch between RTstop/RTmut
    
    sim:
    simulate fastq seq data structures and SNPs.
    options:
        <-ef gencode.gtf> annotation gtf/gff file
        <-gf genome.fa> genome fasta file
        <-o out_prefix> output file prefix
        [-mutf snp.vcf] snp vcf/bed file
        [-sf struct.bed] bed file with structure in 4th column.
        [-alen length] alignment length. (default is 150, must bigger than 0)
        [-rlen length] read fragment length. (default is 300, must bigger than alignment length) (Tips: single end simulation enabled when alignment length is the same as read fragment length)
        [-minalen length] minimum alignment length which sequence lower than this length would be dropped. (default is 30)
        [-faf frequency] enable and set fixed allele frequency. (default is disabled)
        [-genef gene.bed] only the genes in this file can be used in simulation.
        [-bases bases] allow structures show in some bases such as "AC". (default is all bases)
        [-mut] change structure simulation from RTstop to RTmutation.

OutputHeaders

• Here are definitions of headers in output file named (output_dir/file_prefix)_riboSNitch.txt

Field	Description
Chr	Chromosome Name
Position	SNP position in the chromosome (1 based)
Ref	reference snp
Alt	variant snp
Name	dbSNP ID
Score	score of riboSNitch (-1.0 is shown not meaningful)
Strand	strand of this gene
Gene	the longest gene covers this snp
Transcript	the longest transcript covers this snp
Annotation	feature of transcript
Region_in_Genome/Transcriptome	judging region in chromosome/transcript

• Here are definitions of headers in output file named (output_dir/file_prefix)_riboSNitchDetail.txt

Field	Description
Name	dbSNP ID (key with riboSNitch file)
eSDC	eSDC score of riboSNitch
eSDC_pValue	permutation pValue of eSDC score
SNP_Reads_Ratio	proportion of reads that can be divided into specific alleles
Replicate_eSDC	eSDC score of riboSNitch in replicates
Treat_Counts(A|T|C|G)	separate read counts cover snp in treat file(s)
Control_Counts(A|T|C|G)	separate read counts cover snp in control file(s)
Ref_Seq	base sequence of judging region
Ref_Score/Alt_Score	base scores of judging region
StructDiff_pValue	permutation pValues of bases in judging region

License

Licensed GPLv3 for open source use or contact zuoLab (zuozhx@sysucc.org.cn) for commercial use.