DRAGEN v4.4.6 T/N Recipe

The following DRAGEN recipes are specific for Illumina FFPE DNA Prep with Exome 2.5 Enrichment tumor and normal libraries (do not use for tumor only analysis or for a different library prep method). A general recipe for Somatic analysis of Tumor Normal samples with UMI is provided on DRAGEN v4.4 support pages.

/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 
# Inputs
--tumor-fastq-list $PATH                #see 'Input Options' for FQ, BAM or CRAM 
--tumor-fastq-list-sample-id $STRING 
--fastq-list $PATH                      #see 'Input Options' for FQ, BAM or CRAM 
--fastq-list-sample-id $STRING 
# Mapper
--enable-map-align true
--enable-map-align-output true         #save the output BAM (default=false)
--qc-coverage-ignore-overlaps true     #do not double-count overlapping mates
# UMI
--umi-enable true 
--umi-library-type nonrandom-duplex
--umi-min-supporting-reads 1
--umi-start-mask-length 1
--umi-end-mask-length 3
--tumor-normal-has-umi both
# 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
--vc-enable-non-primary-allelic-filter true 
--vc-enable-triallelic-filter false 
--vc-skip-germline-tagging true        
# 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        #see notes below
--variant-annotation-assembly GRCh37/8
# TMB 
--enable-tmb true
# HRD Scoring 
--enable-hrd true                       #requires CNV 
# Microsatellite Instability (MSI) 
--msi-command tumor-only
--max-base-quality 63                                    #default if UMI is enabled
--msi-coverage-threshold 40                              #see notes below 
--msi-microsatellites-file ${microsatellite_file}        #see Resource Files page
--msi-ref-normal-dir ${normal_reference_directory}       #see Resource Files page

Notes and additional options

Hashtable

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

Please see: DRAGEN references

Inputs

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-cram-input $PATH

Mapper

Option	Description
--qc-coverage-ignore-overlaps true	resolve all of the alignments for each fragment and avoid double-counting any overlapping bases

UMI

The above recipe details UMI options specific for the Illumina FFPE DNA Prep with Exome 2.5 Enrichment Library kit.

SNV

The vc-sq-filter-threshold flag can be used to fine-tune the "somatic quality" value at which variants are called in order to balance sensitivity and specificity. Variants with values between the vc-sq-call-threshold and vc-sq-filter-threshold values are labeled as "weak_evidence" in the output VCF file. The default SQ Filter threshold in DRAGEN for tumor-normal analysis is 17.5.

Annotation

Top directory containing Nirvana data file. Instructions on how to download the resource are at https://support.illumina.com/content/dam/illumina-support/help/Illumina_DRAGEN_Bio_IT_Platform_v3_7_1000000141465/Content/SW/Informatics/Dragen/Nirvana_DownloadData_fDG.htm. DRAGEN is expecting a top directory containing the sub-directories Cache, References, Supplementary Annotation.

Microsatellite Instability (MSI)

Microsatellite Instability (MSI) is run in tumor-only mode, but other variants and biomarkers are calculated in tumor-normal mode. msi-coverage-threshold is a required parameter for both tumor-only and tumor-normal, and should be set according to the sample coverage. The sites in the msi-microsatellites-file need to match those in the dist files within the msi-ref-normal-dir .