Somatic-Germline n-of-1 DNAseq Analysis

Call SNV and CNA from somatic and matched germline sample data

How to Setup

Dependencies:

NextFlow, Singularity

Reference Generation:

Run the included download-references.nf

nextflow run brucemoran/somatic_n-of-1/download-references.nf \
  -profile        [str]     singularity,refs
  --assembly      [str]     GRCh37 or GRCh38
  --exomeTag      [str]     naming for exome/panel outputs when supplied; tag is then used in somatic_n-of-1 and batch_somatic main.nf pipelines to select relevant exome reference data

  one of:
  --exomeBedURL   [str]     URL to exome bed file for intervals
  --exomeBedFile  [str]     locally downloaded exome bed file for intervals

General Optional Arguments:

  --exomeAssembly [str]     what assembly is the exome BED based on? (default: GRCH37)
  --cosmicUser    [str]     COSMIC login credentials, user email
  --cosmicPass    [str]     COSMIC login credentials, password

Somatic-Germline n-of-1 Pipeline

About the pipeline:

This pipeline was developed to analyse and report on clinical cancer research. To this end we have tried to make a useful and clinician-readable output. This has been achieved largely on the back of PCGR/CPSR which provide really excellent HTML reports and annotation from multiple clinically relevant sources.

Variant calling uses an 'ensemble' approach with 3 callers (MuTect2, Manta/Strelka2 and Lancet (for exome only)) currently. More may be added in the future. The outputs are combined using our somenone::variantconsensus method. This takes parsed VCF output and combines calls, requiring support from at least 2 callers. It also parses 'raw' (unfiltered) calls, testing those variants that do not have support from 2 callers to see if they are contained in any single callers filtered VCF. In this way we attempt to retain as much true-positive calls as possible.

To run the pipeline:

nextflow run brucemoran/somatic_n-of-1 -profile standard,singularity

N.B. that currently Manta/Strelka2 and Lancet are not available through Conda. Because of this Singularity is required to run the pipeline. This may change and we will update, but Singularity is good so we recommend it anyway.

Arguments:

nextflow run brucemoran/somatic_n-of-1 --help

Formats of --sampleCsv, --sampleCat

To input specific fastq files per samples:

type,sampleID,meta,read1,read2
germline,germ1,whole_blood,/full/path/to/germ1.R1.fastq.gz,/full/path/to/germ1.R2.fastq.gz
somatic,soma1,primary_tumour,/full/path/to/soma1.R1.fastq.gz,/full/path/to/soma1.R2.fastq.gz
somatic,soma2,metastasis,/full/path/to/soma2.R1.fastq.gz,/full/path/to/soma2.R2.fastq.gz

To input directories containing multiple fastqs named {sampleID}*{ext}

type,sampleID,meta,dir,ext
germline,germ1,whole_blood,/full/path/to/dir/with/germ1/fqs,R1.fastq.gz;R2.fastq.gz
somatic,soma1,primary_tumour,/full/path/to/dir/with/soma1/fqs,R1.fastq.gz;R2.fastq.gz
somatic,soma2,metastasis,/full/path/to/dir/with/soma2/fqs,R1.fastq.gz;R2.fastq.gz

The meta column is used for reporting in PCGR, CPSR where sampleID may include clinical/personal data, for example.

Headers of sample.csv file must match above exactly, and you should have only one germline/normal sample per run.

Column type must be germline for one sample only. In case of 2+ germline samples, run with each as germline in turn specifying others as somatic.

####Singularity Hub and containers Unfortunately shub where the containers were housed is gone, so you will need to build the conatiners. We are working on a quay.io repo. Please pull our github Singularity repo (brucemoran/Singularity) and use our recipe_builder.sh script:

git clone https://github.com/brucemoran/Singularity
cd Singularity
sudo sh ./utilities/recipe_builder.sh <recipe_name> <$NXF_SINGULARITY_CACHE> shub

This builds a correctly named container into the $NXF_SINGULARITY_CACHE so the pipeline will run.