DRAGEN v4.3 Recipe

The following DRAGEN recipes are specific for Illumina FFPE DNA Prep with Exome 2.5 Enrichment libraries. A general recipe for Somatic analysis of Tumor Normal samples with UMI is provided on DRAGEN v4.3 support pages.

The workflow is a multi-phase process. Before DRAGEN v4.4, the somatic UMI Tumor/Normal pipeline can only process pre-collapsed BAM files. To use this pipeline with raw FASTQ files, individually map/align & read collapse the tumor and normal samples. The resulting BAM files are then fed into the variant calling step.

Preprocessing required for UMI T/N Variant Calling

Run mapping and UMI collapsing on each of the samples:

/opt/dragen/$VERSION/bin/dragen        #DRAGEN install path 
--ref-dir $REF_DIR                     #path to DRAGEN linear hashtable 
--output-directory $OUTPUT 
--intermediate-results-dir /staging    #tmp dir on fast SDD 
--output-file-prefix $PREFIX 
--fastq-list $PATH                     #see 'Input Options' below for FQ, BAM or CRAM 
--fastq-list-sample-id $STRING
--enable-map-align true
--enable-map-align-output true         #save the output BAM 
--qc-coverage-ignore-overlaps true     #do not double-count overlapping mates
--umi-enable true 
--umi-library-type nonrandom-duplex
--umi-metrics-interval-file $VC_TARGET_BED
--umi-min-supporting-reads 1
--umi-start-mask-length 1
--umi-end-mask-length 3

Variant Calling Step

Run variant calling on a tumor/normal pair:

/opt/dragen/$VERSION/bin/dragen        #DRAGEN install path  
--ref-dir $REF_DIR                     #path to DRAGEN linear hashtable 
--output-directory $OUTPUT 
--intermediate-results-dir /staging    #tmp dir on fast SDD 
--output-file-prefix $PREFIX 
--tumor-bam-input $TUMOR_BAM           #from preprocessing steps 
--bam-input $BAM                       #from preprocessing steps 
# Mapper 
--enable-map-align false
# Small variant caller 
--enable-variant-caller true 
--vc-target-bed $VC_TARGET_BED         #see notes below
--vc-systematic-noise $PATH            #see Resource Files  
--vc-enable-umi-solid true 
--vc-sq-call-threshold 3 
--vc-sq-filter-threshold 15         
# CNV 
--enable-cnv true
--cnv-use-somatic-vc-baf true
--cnv-target-bed $VC_TARGET_BED 
--cnv-combined-counts $PATH            #see Resource Files 
# Annotation                           #annotation is required if enabling TMB
--enable-variant-annotation true 
--variant-annotation-data $PATH        #see notes below
--variant-annotation-assembly GRCh37/8 
# TMB 
--enable-tmb true 

Notes and additional options

Hashtable

For DRAGEN somatic runs it is recommended to use the linear (non-graph) hashtable.

Please see: DRAGEN references

Input options for Preprocessing

DRAGEN input sources include: fastq list, fastq, bam, or cram. For preprocessing, whether the sample is tumor or normal does not need to be distinguished (although it can be) because the output is simply a collapsed BAM file. Here are support pages for the formats of different input file types.

FQ list Input

--fastq-list $PATH 
--fastq-list-sample-id $STRING 

or

--tumor-fastq-list $PATH 
--tumor-fastq-list-sample-id $STRING 

FQ Input

--fastq-file1 $PATH 
--fastq-file2 $PATH 
--RGSM $STRING 
--RGID $STRING 

or

--tumor-fastq1 $PATH 
--tumor-fastq2 $PATH 
--RGSM-tumor $STRING 
--RGID-tumor $STRING 

BAM Input

--bam-input $PATH

or

--tumor-bam-input $PATH

CRAM Input

--cram-input $PATH

or

--tumor-bam-input $PATH