BaseSpace Sequence Hub (BSSH)

DRAGEN Somatic App Version 4.3.17 in BaseSpace Sequence Hub is used to analyze sequencing data from llumina FFPE DNA Prep with Exome 2.5 Enrichment libraries. Refer to online support for general information about using the DRAGEN Somatic app. The following settings are specific for Illumina FFPE DNA Prep with Exome 2.5 Enrichment libraries.

Access to Resource Files

The analysis workflow uses resource files. See the Resource Files page for details on where to find those files, which should be manually uploaded to BSSH and available for analyses.

Analysis Settings

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Open the DRAGEN Somatic App

Version 4.3.17

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Input Data

Select Biosamples or Samples as the input type to launch the analysis.

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Configuration

• Analysis name—Name of the analysis

• Save Result To—Project to store analysis results in.

• Pipeline Configuration—Select Somatic Small Variant Caller (Aligned BAM or CRAM input).

• Somatic CNV Calling—Select Tumor-Normal

• Input FASTQ—Select the TUMOR FASTQ and the paired NORMAL FASTQ, and set the Sample Sex to Unknown. Click Add a New Row to input another tumor-normal pair. Each pair is entered as a new row.

• Reference—The reference genome to use for alignment, and the same Reference used above in the Map/Align step. Currently Homo sapiens [UCSC] hg19 v4 and Homo sapiens [1000 Genomes] hg38 v4 are supported somatic for variant calling.

• Targeted Regions—The genomic regions targeted for enrichment, and the same Targeted Regions used above in the Map/Align step. If no spike-in probes were included in enrichment, select Twist Bioscience for Illumina Exome 2.5 Plus Panel. If other spike-in probes were included in enrichment, select Custom BED and under Target BED File select Select Dataset File(s). This must be a BED file of the combined coverage areas, ie., Exome 2.5 Plus Panel and the custom panel.

• Systematic Noise Filter—Leave Enable Systematic Noise Filter checked. Select Dataset File(s) and choose the systematic noise file that matches the Reference genome used for alignment.

• CNV Baseline—Select Custom (Select CNV Baseline Files below). Select Dataset File(s). Files must be of the same type (eg, all .target.counts or all .target.counts.gc-corrected.). Navigate to the Project containing the target.counts files and select those that match the Reference genome selected above and has the desired handling of GC correction.

• GC Bias Correction—The type of CNV Baseline Files must match the checkbox here. For instance, if .target.counts.gc-corrected are used for the CNV Baseline Files, then keen GC Bias Correction enabled.

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Biomarkers

• View the Biomarkers options by clicking on + Biomarkers

• Enable HRD scoring—Check

CNV calling must be enabled in order to calculate HRD.

• Tumor Mutational Burden—Check

TMB will be calculated using WES coding regions for the selected genome reference, so Custom TMB Regions do not need to be supplied.

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• Microsatellite Instability—Check

It is recommended to run the tumor sample in tumor-only mode for highest performance of this biomarker. Note that a flag will be added to Additional Argument, a setting that allows MSI to be calculated in tumor-only mode while other analyses are calculated in tumor-normal mode. This biomarker is under active development.

• MSI References—Select Dataset File(s) and select the individual distance files of the normal reference samples (hg*-WES-FFPE-*.microsat_normal.dist)

• Custom MSI Regions—Select Dataset File(s) and select the microsatellite list that maches the reference genome used for alignment: WES_v1.1.0_hg*_microsatellites.list

Structural Variant (SV) caller results have not been evaluated for accuracy. Calculations for SV can add significant runtime to the analysis.

UMI Settings

• View the UMI Settings options by clicking on + UMI Settings

• Enable UMI—Check

• UMI Library Type—Nonrandom-duplex

• UMI-Aware Variant Calling—Low depth

• UMI Min Supporting Reads—1 (Change from the default of 2)

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Advanced Settings

• View the Advanced Settings options by clicking on + Advanced Settings

• Enable Duplicate Marking—Uncheck this setting.

• Enable Common Germline Variant Tagging—Uncheck this setting.

• Somatic Quality Filtering—Check Enable Setting Somatic Quality Filtering Thresholds. Set Somatic variant quality call threshold to 3 and Somatic variant quality filter threshold to 15.

• Nirvana Annotation—Check Enable Nirvana variant annotation.

Variant Annotation must be enabled if calculating TMB.

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Additional Arguments

• View the Additional Arguments options by clicking on + Additional Arguments

• Check the box acknowledging the warning around using command-line arguments

• Include these flags as Additional DRAGEN Command-line Arguments --umi-start-mask-length 1 --umi-end-mask-length 3 --msi-command tumor-only --qc-coverage-ignore-overlaps true

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Click Launch Application