CQDG denovo pipelines

Introduction

🚧 WIP : this pipeline recombine gvcf for family's samples in order to facilitate denovo identification.

(Include Damien's workflow schema?)

Usage

Samples

The workflow will accept sample data in two format (called V1 and V2). The path to the sample file must be specified with the "sampleFile" parameter.

1. The first format is used by default and looks as follows:

sampleV1.tsv

FAMILY_ID           Patient1_File        Patient2_File        Patient3_File

CONGE-XXX       CONGE-XXX-01.hard-filtered.gvcf.gz   CONGE-XXX-02.hard-filtered.gvcf.gz   CONGE-XXX-03.hard-filtered.gvcf.gz
CONGE-YYY       CONGE-YYY-01.hard-filtered.gvcf.gz   CONGE-YYY-02.hard-filtered.gvcf.gz   CONGE-YYY-03.hard-filtered.gvcf.gz

2. The second format is used in older data and includes the sequencing type (WGS or WES)

sampleV2.tsv

FAMILY_ID           SEQUENCING_TYPE          Patient1_File        Patient2_File        Patient3_File

CONGE-XXX       WES       CONGE-XXX-01.hard-filtered.gvcf.gz   CONGE-XXX-02.hard-filtered.gvcf.gz   CONGE-XXX-03.hard-filtered.gvcf.gz
CONGE-YYY       WES       CONGE-YYY-01.hard-filtered.gvcf.gz   CONGE-YYY-02.hard-filtered.gvcf.gz   CONGE-YYY-03.hard-filtered.gvcf.gz

The file format can be chosen with the "sampleFileFormat" parameter (either "V1" or "V2", default "V1"). Note that both types are tab-delimited (.tsv)

Next, if the file format is "V1", the sequencing type can be specified with the "sequencingType" parameter (either "WGS" for Whole Genome Sequencing or "WES" for Whole Exome Sequencing, default "WGS")

Note

The sequencing type also determines the type of variant filtering the pipeline will use.

In the case of Whole Genome Sequencing, VQSR (Variant Quality Score Recalibration) is used (preferred method).

In the case of Whole Exome Sequencing, Hard-filtering needs to be used.

References

Reference files are necessary at multiple steps of the workflow, notably for joint-genotyping,the variant effect predictor (VEP) and VQSR.

Specifically, we need a reference genome directory and filename specified with the referenceGenome and referenceGenomeFasta parameters respectively.

⚠️ (TO DO: I think these two parameters could be combined, I don't think we use any other file than the Fasta in the referenceGenome directory.) ⚠️

Generally, we use the Homo_sapiens_assembly38.fasta as referenceGenome (see Resources)

Next, we also need broader references, which are contained in a path defined by the broad parameter.

The broad directory must contain the following files:

• The interval list which determines the genomic interval(s) over which we operate: filename of this list must be defined with the intervalsFile parameter
• Highly validated variance ressources currently required by VQSR. These are currently hard coded in the pipeline!
  • HapMap file : hapmap_3.3.hg38.vcf.gz
  • 1000G omni2.5 file : 1000G_omni2.5.hg38.vcf.gz
  • 1000G reference file : 1000G_phase1.snps.high_confidence.hg38.vcf.gz
  • SNP database : Homo_sapiens_assembly38.dbsnp138.vcf.gz

Finally, the vep cache directory must be specified with vepCache, which is usually created by vep itself on first installation. Generally, we only need the human files obtainable from https://ftp.ensembl.org/pub/release-112/variation/vep/homo_sapiens_vep_112_GRCh38.tar.gz

Stub run

The -stub-run option can be added to run the "stub" block of processes instead of the "script" block. This can be helpful for testing.

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Parameters summary

Parameter name	Required?	Accepted input
sampleFile	Required	file
sampleFileFormat	Optional	V1 or V2, default V1
sequencingType	Optional	WGS or WES, default WGS
referenceGenome	Required	path
referenceGenomeFasta	Required	file
broad	Required	path
intervalsFile	Required	list of genome intervals
vepCache	Required	path

Pipeline Output

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Resources

The documentation of the various tools used in this workflow are available here:

Nextflow

bcftools

GATK:

• CombineGVCFs
• GenotypeGVCFs
• VariantRecalibrator
• VariantFiltration)

VEP

Reference files 🚧