GTN_sWGS

Description

This is a pipeline to obtain relative copy number from fastq files from sWGS. This pipeline is written using nextflow. I have aligned here to hg19 for ease, as hg38 bin annotations are not easily available for analysis with QDNASeq at this time.

Pipeline summary

Processes

1. FASTQC_RAW
   • fastQC is used on the raw files to determine their quality
2. TRIMMOMATIC
   • Trimmomatic is used to trim out adapters and polyG tails. These are available in a text file in /bin and specified with params.adap
3. FASTQC_TRIM
   • Used to determine the quality of the sequences after trimming out adapters and polyG tails.
4. MultiQC_TRIM
   • Used to collect results from fastQC before and after trim and create one report for all fastqc reports
5. ALIGN
   • bwamem and samtools are used to align the sWGS files to hg19 and sort/index resulting bam files. A genome index is needed for this step and its path is specified using params.index. qualimap is also used to determine efficiency of the mapping
6. QDNNASeq is used to obtain relative copy number from bam files using a customised Rscript in $projectDir/bin. This process is a little different, as two separate scripts are needed depending on whether the sex chromosomes are to be included in analysis as specified using the --sexChrIncl flag:
   • QDNASEQ - default option. Sex chromosomes are not included in analysis, nextflow directs to the QDNASeq.R script in $projectDir/bin
   • QDNASEQSEX - Sex chromosomes included in analysis, based on the presence of the --sexChrIncl flag in the main nextflow run command

Parameters

• --reads can be used to specify the path to the reads. Default is set to $projectDir/sWGS/fastq/*_{R1,R2}.fq.gz
• --index path to BWA indexed genome. Default is hg19 in reference data in ICR cluster Alma.
• --outdir where to publish results. Deafult $projectDir/results
• --adap path to textfile where adapter sequences are. These are essential for trimmomatic.
• --binsize default 30, allows you to specify the bin size for QDNASeq. available options are 1, 5, 10, 15, 30, 50, 100, 500, and 1000 kbp.
• --sexChrIncl specify this flag if you want to include sex chromsomes. Othersie QDNASeq will ignore them.

QDNASeq

Two customised scripts for QDNASeq can be found in /bin (QDNAseq.R QDNAseq_sexChrIncl.R). These have been made using the QDNASeq vignette available here

Run it

The main script is in 8-GTN_sWGS.nf. You will also need bin/QDNAseq.R and bin/QDNAseq_sexChrIncl.R which must be executable, use chmod +x to do this.

Depending on the cluster, the way the main script is executed will be different. nextflow.config was made to be compatible with ICR alma.