khandaud15 / RNA-Seq-Variant-Calling

Snakemake based workflow for calling Variants on RNA-Seq data in a joint calling mode

RNA-Seq Variant Calling Pipeline

This workflow is based on calling variants on RNA-Seq data using GATK4. the pipeline starts all the way from raw Fastq files and end up with VCF file with the joint calling.

Main Steps

Mapping to the Reference

Tools involved:STAR

The pipeline begin with mapping RNA reads to a reference, we have used STAR aligner because it increased sensitivity compared to other alligner(especially for INDELS), as well as use STAR’s two-pass mode to get better alignments around novel splice junctions.

Add read groups, sort, mark duplicates, and create index using Picard and Samtools

The Star Mapping step produces a BAM/SAM file, which we then put through the usual Picard processing steps: adding read group information, sorting, marking duplicates and indexing for downstream processing.

Split'N'Trim and Reassign mapping qualities

Tools Involved:SplitNCigarReads

This step splits reads into exon segments (getting rid of Ns but maintaining grouping information) and hard-clip any sequences overhanging into the intronic regions as well reassign mapping qualities to the alligned reads because STAR Napping assigns good alignments a MAPQ of 255 (which technically means “unknown” and is therefore meaningless to GATK)

Base Quality Recalibration

Tools involved: BaseRecalibrator, Apply Recalibration, AnalyzeCovariates

This step correct any systematic bias observed in the data. These Biases can originate from biochemical processes occured during library preparation and sequencing, from manufacturing defects in the chips, or instrumentation defects in the sequencer. The recalibration step involves collecting covariate statistics from all base calls in the dataset, building a model from those statistics, and applying base quality adjustments to the dataset based on the resulting model.

Variant Calling

Tools involved: HaplotypeCaller

The step calls the SNPs and indels simultaneously via local de-novo assembly of haplotypes in an active region

Required Tools

• FastQC (A quality control tool for high throughput sequence data)

• Trim-galore (Automates quality control and adapter trimming of fastq files)

• STAR (Spliced aware ultrafast transcript alligner to refernece genome)

• Picard (Cammand line set tool to manipulate high-throughput sequencing data)

• Samtools (Tool for manipulating alignments in the SAM/BAM format, including sorting, merging, indexing and generating alignments in a per-position format)

• GATK4 (software package to covers all major variant classes from NGS Dataset)

Index the genome for 1st pass allignment, the 2nd pass allignemnt uses the new index from merged SJ.out.tab files from the script

 STAR  --runMode genomeGenerate --runThreadN 24 --genomeDir ./ --genomeFastaFiles hg38.fa   --sjdbGTFfile gencode.v30.annotation.gtf 

To Run the pipeline on cluster using this command 'modify cluster.json parameters according to your cluster configuration

snakemake -j 999 --configfile config.yaml --use-conda --nolock --cluster-config cluster.json --cluster "sbatch -A {cluster.account} -p {cluster.partition}  -c {cluster.ncpus} -n {cluster.ntasks }  -t {cluster.time} --mem {cluster.mem}"