Whole-genome Sequencing Data Variant Calling

Summary of methods

• Index generated with BWA
• Filter and trim reads with fastp
• Strand determination with how are we stranded here
• Quality checking of reads with FastQC
• Reads mapped to reference BWA MEM
• Alignment post-processing and QC with Picard
• Variant calling with bcftools
• Variant filtering with GATK
• Aggregation of QC tables using MultiQC

Parameters

Required parameters:

• --pubdir

  • Default: /<PATH>
  • Description: The directory that the saved outputs will be stored.

• -w

  • Default: /<PATH>
  • The directory that all intermediary files and nextflow processes utilize. This directory can become quite large. This should be a location on scratch space or other directory with ample storage.

• --sample_folder

  • Default: /<PATH>
  • The path to the folder that contains all the samples to be run by the pipeline. The files in this path can also be symbolic links.

• --fasta

  • Default: /<PATH>
  • Path to the reference genome in FASTA format

• --read_type

  • Default: PE
  • Comment: Type of reads: paired end (PE) or single end (SE).

Optional parameters:

General optional parameters:

• --concat_lanes

  • Default: false
  • Options: false and true. Default: false. If this boolean is specified, FASTQ files will be concatenated by sample. Used in cases where samples are divided across individual sequencing lanes.

• --extension

  • Default: .fastq.gz
  • Expected file extension for input read files, modify if files are not compressed or have a different form (e.g. ".fastq" or ".fq.gz")

• --pattern

  • Default: *_R{1,2}*
  • Expected R1/R2 matching pattern for paired-end read files in the --sample_folder path. In concert with --extension the default values will match files such as sampleID.L001.R1.fastq.gz and sampleID.L001.R2.fastq.gz

• keep_intermediate

  • Default: false
  • If true workflow will output intermediate alignment files (unsorted BAMs, etc.)

• keep_reference

  • Default: false
  • If true workflow will save a copy of the BWA indices to the output directory

fastp filtering parameters:

• --quality_phred

  • Default: 15
  • Quality score threshold.

• --unqualified_perc

  • Default: 40
  • Percent threshold of unqualified bases to pass reads.

• --tmpdir

  • Default: "~/scratch/$USER/tmp/"

BWA mem mapping parameters:

• --mismatch_penalty

  • Default: "-B 8"

• --bwa_min_score

  • Default: null
  • Threshold of alignment quality score to emit read alignment

Variant calling parameters:

• --ploidy

  • Default: 2
  • Options: 1, 2

• --mpileup_depth

  • Default: 100
  • For a given position, read maximally INT reads per input file

• --skip_indels

  • Default: false
  • If true, do not call indel sites

• --variants_only

  • Default: true
  • If true, only emit variant sites (e.g. not homozygous reference)

Variant filtering parameters:

• --filter_dp

  • Default: "DP < 25"
  • Sites with depth lower than this value will have a filter flag set as "LowCoverage"

• --filter_very_low_qual

  • Default: "QUAL < 30.0"
  • Sites with a quality score less than this value will have a filter flag set as "VeryLowQual"

• --filter_low_qual

  • Default: "QUAL > 30.0 && QUAL < 50.0"
  • Sites with a quality score less than this value will have a filter flag set as "LowQual"

• --filter_qd

  • Default: "QD < 1.5"
  • Sites with a depth-normalized quality score less than this value will have a filter flag set as "LowQD"

• --filter_fs

  • Default: "FS > 60.0"
  • Sites with a strand bias greater than this value will have a filter flag set as "StrandBias"

Pipeline Default Outputs

Naming Convention	Description
wgs_cohort_variants_bcftools_filtered.vcf	Soft-filtered VCF including all samples
wgs_cohort_variants_bcftools_filtered_genotype_table.tsv	RSEM-generated gene-level TPM counts merged across all samples
wgs_cohort_variants_bcftools_filtered_site_table.tsv	RSEM-generated isoform-level raw counts merged across all samples
wgs_report.html	Nextflow autogenerated report
index/	BWA-generated indices saved with optional parameter keep_reference
multiqc/	MultiQC report summarizing quality metrics across all samples in the run
${sampleID}/stat/	QC and Picard metrics files for each sample
trace.txt	Nextflow trace of processes

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Last update: January 12, 2025