Germline WES

DRAGEN Recipe - Germline WES

Overview

This recipe is for processing whole exome sequencing data for germline workflows.

Example Command Line

For most scenarios, simply creating the union of the command line options from the single caller scenarios will work.

• Configure the INPUT options

• Configure the OUTPUT options

• Configure MAP/ALIGN depending on if realignment is desired or not

• Configure the VARIANT CALLERs based on the application

• Configure any additional options

• Build up the necessary options for each component separately, so that they can be re-used in the final command line.

We highly recommend using a pangenome reference for human samples (excluding RNA). For more details, refer to Dragen Reference Support.

The following are partial templates that can be used as starting points. Adjust them accordingly for your specific use case.

#!/bin/bash
set -euo pipefail

# Path to DRAGEN hashtable
DRAGEN_HASH_TABLE=<REF_DIR> # pangenome reference for human samples

# Path to output directory for the DRAGEN run
OUTPUT=<OUT_DIR>

# File prefix for DRAGEN output files
PREFIX=<OUT_PREFIX>

# Define the input sources, select fastq list, fastq, bam, or cram.
INPUT_FASTQ_LIST="
  --fastq-list $FASTQ_LIST \
  --fastq-list-sample-id $FASTQ_LIST_SAMPLE_ID \
"

INPUT_FASTQ="
  --fastq-file1 $FASTQ1 \
  --fastq-file2 $FASTQ2 \
  --RGSM $RGSM \
  --RGID $RGID \
"

INPUT_BAM="
  --bam-input $BAM \
"

INPUT_CRAM="
  --cram-input $CRAM \
"

# Select input source, here in this example we use INPUT_FASTQ_LIST
INPUT_OPTIONS="
  --ref-dir $DRAGEN_HASH_TABLE \
  $INPUT_FASTQ_LIST \
"

OUTPUT_OPTIONS="
  --output-directory $OUTPUT \
  --output-file-prefix $PREFIX \
"

MA_OPTIONS="
  --enable-map-align true \
  --enable-sort true \
  --enable-duplicate-marking true \
"

CNV_OPTIONS="
  --enable-cnv true \
  --cnv-target-bed $CNV_TARGET_BED \
  --cnv-combined-counts $CNV_PANEL_OF_NORMALS \
"

SNV_OPTIONS="
  --enable-variant-caller true \
  --vc-target-bed $VC_TARGET_BED \
"

SV_OPTIONS="
  --enable-sv true \
  --sv-exome true \
  --sv-call-regions-bed $SV_TARGET_BED \
"

HLA_OPTIONS="
--enable-hla=true \
--hla-enable-class-2=true \ # only if the panel has sufficient coverage for class II HLA typing 
"

# Construct final command line
CMD="
  dragen \
  $INPUT_OPTIONS \
  $OUTPUT_OPTIONS \
  $MA_OPTIONS \
  $CNV_OPTIONS \
  $SNV_OPTIONS \
  $SV_OPTIONS \
  $HLA_OPTIONS \
"

# Execute
echo $CMD
bash -c $CMD

Additional Notes and Options

Optional settings per component are listed below. Full option list at this page.

CNV

Please include the matched normal sample in the CNV panel of normals.

Generating Panel of Normals (PON)

WES CNV requires PON files. Follow the two steps below to generate CNV PON:

1. Target counts generation (per normal sample): Target counts of individual normal sample should be generated as baseline. Any options used for panel of normals generation (BED file, GC Bias Correction, etc) should be matched when processing the case sample.

CNV_PON_OPTIONS="
  --enable-cnv true \
  --cnv-target-bed $CNV_TARGET_BED \
"

CMD="
  dragen \
  $INPUT_OPTIONS \
  $OUTPUT_OPTIONS \
  $CNV_PON_OPTIONS \
"

1. Combined counts generation: Individual PON counts can be merged into a single file as a <prefix>.combined.counts.txt.gz file.

CNV_COMBINED_COUNTS_OPTIONS="
  --enable-cnv true \
  --cnv-generate-combined-counts true \
  --cnv-normals-list $CNV_NORMALS_LIST \
"

CMD="
  dragen \
  $INPUT_OPTIONS \
  $OUTPUT_OPTIONS \
  $CNV_COMBINED_COUNTS_OPTIONS \
"

$CNV_NORMALS_LIST is a single text file with paths to each target counts file generated by step1 (either .target.counts.gz or .target.counts.gc-corrected.gz). Output will have a PON file with suffix .combined.counts.txt.gz file. Use the PON file in case sample runs of DRAGEN CNV with --cnv-combined-counts option.

For more information, see Panel of Normals.

SNV

Note that we do not recommend changing the default QUAL thresholds of 3 for DRAGEN-ML and 10 for DRAGEN without ML. These values differ from each other because DRAGEN-ML improves the calibration of QUAL scores, leading to a change in the scoring range (see QUAL, QD, and GQ Formulation).

HLA