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 #see Resource Files page
--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 Resource Files page
--vc-systematic-noise $PATH            #see Resource Files page
--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 page
# Annotation                           #annotation is required if enabling TMB
--enable-variant-annotation true
--variant-annotation-data $PATH
--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

Additional Biomarkers

Microsatellite Instability (MSI) and Homologous Recombination Deficiency (HRD) have also been evaluated for samples with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment protocol. See Additional Information on determining these biomarkers.