Pipeline to analyse Whole Genome Sequencing (WGS) data

tested in C elegans dataset

This project consists of a pipeline to analyse whole genome data.

Fastq files that I used for testing are obtained from the work entitled: "Optogenetic mutagenesis in Caenorhabditis elegans" of Kentaro Noma and Yishi Jin from San Diego University (1) Thanks to these authors for their very interesting data!!

Characteristics of the dataset

The organism (C elegans) is diploid. Reads are paired-end, obtained from Illumina technology. Read length is 90bp

BEFORE anything:

Read entirely my pipeline wholeGenworkflow.sh and verify that it serves your purpose.

A) Preparing Directories

This tree (files and folders hierarchy) allows to work comfortably with this pipeline:

home/johanna/WGS 
├── genomesref
│   ├── species.project.release.annotations.gff3.gz
│   ├── species.project.release.genomic.fa.gz
│   └── derived files (indexes, reference vcf, etc)
├── OPTOCelegans
│   ├── fastq
│   │   └── every_fastq_file.gz
│   ├── QC
│   │   ├── every_fastqc.html
│   │   └── every_fastqc.zip
│   ├── TRIMM
│   │   └── trimming_results
│   ├── ALIGNED
│   │   └── alignement_results (sam, bam, FINAL.vcf, etc)
│   └── SRR_Acc_List.txt
├── ReadMe_personalProject
├── wholeGenworkflow.sh
└── other scripts(.sh,.py,.r,etc) if neccessary

I named working directory as "WGS". Feel free to change it i.e. "wholeGenSeq" or other coherent name with the technical context.

These directories MUST exist before using the pipeline, even if zero files within:

WGS
genomesref
OptoCelegans
- fastq

In next steps we will fill them, and create more, using the pipelines. I strongly recommend to fit to this same tree structure in your computer. You can also clone this work :

git clone https://gitlab.com/JohannaGL/wgs.git

"OPTOCelegans" is the project here. You can add other projects and/or other genomesref, that's why the pipelines are not inside the project, so we can re-use them.

If working with other diploid species, be aware that VCF generator provided here "gff2vcf_Celegans.py" only works for the release W260 of C elegans.

A) Preparing fastq files

If you have your own fastq files:
- put them in 'WGS/OPTOCelegans/fastq'
If you dont have them:
1. download Sra-ToolKit from NCBI, unzip.
2. next, in a code or text editor, open fastqFromSRA provided here, change paths by yours and save.
3. then, download the *SRR_Acc_List.txt" (from SRA NCBI) to location as shows the tree
4. Finally, run fastqFromSRA. It oma K, Jin Y. Optogenetic mutagenesis in Caenorhabditis elegans. Nat Commun. 2015 Dec 3;6:8868. doi: 10.1038/ncomms9868. PMID: 26632265; PMCID: PMC4686824.works for paired end data. Run it in your linux shell, from the working directory:

~ $ cd WGS
~/WGS $ ./fastqFromSRA.sh

B) Using 'wholeGenworkflow' pipeline

Open wholeGenworkflow.sh in a code editor or in gedit.
Change the paths by yours
run
The pipeline asumes you have already in your system all the tools and software:
- bwa
- samtools
- trimmomatic
- picard
- GATK4 (v 4.1.3.0) ==> I tested only with this version !
- (eventually bcftools if reference VCF is created on the fly)

References

Noma K, Jin Y. Optogenetic mutagenesis in Caenorhabditis elegans. Nat Commun. 2015 Dec 3;6:8868. doi: 10.1038/ncomms9868. PMID: 26632265; PMCID: PMC4686824.

Other sources

https://gatkforums.broadinstitute.org/gatk/discussion/44/base-quality-score-recalibration-bqsr https://www.biostars.org/p/281776/ https://github.com/gatk-workflows/gatk4-data-processing/blob/master/processing-for-variant-discovery-gatk4.wdl

author of this pipeline:

Johanna GALVIS - 2019

johaGL / wgs