Demo Data is available on BSSH with the Project name "NovaSeq6000/NovaSeqX: Illumina FFPE DNA Prep with Exome 2.5 Enrichment - Demo Data" and on ICA with the Bundle name "Illumina FFPE DNA Prep with Exome 2.5 Enrichment - Demo Data".

Data includes 14 samples, or seven Tumor/Normal pairs:

1. SNV-tumor-AF10-NovaSeq6K & SNV-normal-NovaSeq6K: Seracare Seraseq Tumor Mutation DNA Mix v2 AF10 and Seraseq WT (DNA/RNA) Reference Material. Samples with known truth variants at expected Variant Allele frequency at 10%, sequenced on the NovaSeq6000 Sequencing System.

2. SNV-tumor-AF5-NovaSeqX & SNV-normal-NovaSeqX: Seracare Seraseq Tumor Mutation DNA Mix v2 AF5 and Seraseq WT (DNA/RNA) Reference Material. Seracare Reference Material diluted to expected Variant Allele frequency at 5%, sequenced on the NovaSeqX Sequencing System.

3. CNV-tumor-NovaSeq6K & CNV-normal-NovaSeq6K: Seracare Seraseq Solid Tumor CNV Mix +3 Copies and Seraseq WT (DNA/RNA) Reference Material. Sample with known CNV events at expected duplication events with fold change at 3x, sequenced on the NovaSeq6000 Sequencing System.

4. FFPE-tumor-21706-NovaSeq6K & FFPE-normal-21707-NovaSeq6K: clinical formalin-fixed paraffin-embedded (FFPE) tumor sample 21706 and benign adjacent tissue sample 21707. FFPE samples sequenced on the NovaSeq6000 Sequencing System.

5. FFPE-tumor-21706-NovaSeqX & FFPE-normal-21707-NovaSeqX: clinical formalin-fixed paraffin-embedded (FFPE) tumor sample 21706 and benign adjacent tissue sample 21707. FFPE samples sequenced on the NovaSeqX Sequencing System.

6. FFPE-tumor-12293-exome-NovaSeq6K & FFPE-normal-12294-exome-NovaSeq6K: FFPE tumor sample 12293 and benign adjacent tissue sample 12294, enriched with the Twist Exome v2.5 panel. FFPE samples sequenced on the NovaSeq6000 Sequencing System.

7. FFPE-tumor-12293-exome-plus-spike-NovaSeq6K & FFPE-normal-12294-exome-plus-spike-NovaSeq6K: FFPE tumor sample 12293 and benign adjacent tissue sample 12294, enriched with the Twist Exome v2.5 panel and a custom spike-in panel. FFPE samples sequenced on the NovaSeq6000 Sequencing System.

Sample pair #7 (FFPE 12293/12294 enriched with and without a custom spike-in panel) was not included in variant calling because a systematic noise file and a Panel of Normals were not available for these combined covered regions. The pre-built WES systematic noise file based on the exome region (without spike-in) can be used, but precision in the custom spike-in areas may be reduced. The pre-built PON target counts based on the exome region (without spike-in) can be used, but CNV events may be inaccurate. Differences in coverage can seen by viewing the bam files.

Distributed as a BaseSpace Project and an ICA Bundle

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.

Input options for Preprocessing

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.

HRD is under development. Performance improvements are planned with upcoming software releases. In the meanwhile, HRD results should be considered preliminary. HRD can be enabled by adding --enable-hrd true to a command-line or checking the Enable HRD in BaseSpace and ICA applications. Note that CNV calling must be enabled in order to calculate HRD.

MSI is under active development.

In upcoming software releases, it will be possible to calculate MSI in Tumor/Only mode even while running a Tumor/Normal workflow, thus eliminating the need for an additional analysis.

DRAGEN command-line recipe

/opt/dragen/$VERSION/bin/dragen
--msi-command tumor-only
--max-base-quality 63
--msi-microsatellites-file ${microsatellite_file}  
--msi-ref-normal-dir ${normal_reference_directory} 
--ref-dir $REF_DIR                      #path to DRAGEN linear hashtable                
--output-directory $OUTPUT   
--output-file-prefix $PREFIX 
--tumor-bam-input $TUMOR_BAM            #from preprocessing steps 
--enable-map-align false

BSSH Somatic App

Configuration

• Somatic CNV Calling—Select Tumor-only

• Input BAM—Select the tumor BAM and the Sample Sex to Unknown. Click Add a New Row to input another tumor sample. Each tumor BAM is entered as a new row.

• Systematic Noise Filter—uncheck Enable Systematic Noise Filter.

• CNV Baseline—None

Biomarkers

• View the Biomarkers options by clicking on + Biomarkers

• Microsatellite Instability—check Enable MSI calling

• MSI References—navigate to the Project containing the reference directory of *microsat_normal.dist files that match the reference genome used.

• Custom MSI Regions—navigate to the *.tsv file containing the microsatellite list file that matches the reference genome used.

Advanced Settings

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

• Enable Duplicate Marking—Uncheck this setting

• Enable Variant Calling—Uncheck this setting

• Enable Common Germline Variant Tagging—Uncheck this setting

• Enable Multi-allelic Filtering—Uncheck this setting

Additional Arguments

• Check the acknowledge and agree box

• In Additional DRAGEN Command-line Arguments, add --enable-cnv false

ICA

Input Files

• Enter the Tumor BAM File for tumor sample(s)

• Enter the Tumor BAM Index for the tumor sample(s)

• Systematic Noise BED File—Do not select

• CNV Files—Do not select

• Microsatellites File—navigate to the *.list file containing the microsatellite list file that matches the reference genome used.

• Microsatellites Normal References Directory—navigate to the directory of *microsat_normal.dist files that match the reference genome used.

Variant Calling Options

• Enable Small Variant Caller—false

• Enable Germline Tagging—false

Targeted Callers

• MSI Command—tumor-only

• MSI Coverage Threshold—60

• Enable Tumor Mutational Burden—false

Variant Annotation Options

Enable Variant Annotation—false

llumina FFPE DNA Prep with Exome 2.5 Enrichment is part of an integrated whole-exome sequencing (WES) Tumor-Normal workflow to deliver variant calling and biomarker analysis in low-input formalin-fixed paraffin-embedded (FFPE) samples. This page provides software user guides to Illumina's cloud-based and on-premise solutions for the data analysis of this library preparation kit.

The software performs the following workflows to analyze sequencing data.

• DNA Mapping and Aligning

• Somatic Small Variant (SNV) Caller

• Copy Number Variant (CNV) Caller

• Tumor Mutational Burden (TMB)

• Variant annotation

Notes on additional biomarkers:

Homologous Recombination Deficiency (HRD) is also under development, and performance improvements are expected with the release of upcoming software versions.

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

Exploring germline variants that might play a role in tumor analysis

Will enabling germline tagging in T/N workflows provide germline variants in the SNV/INDEV VCF?

Only those somatic variants that match a known allele from a database. In T/N mode, where the VCF usually includes only somatic variants, then it is possible that some of these somatic variants may match a known allele from a database, and then be tagged as germline.

Germline variants can be labeled in the somatic VCF by adding a flag to the command line recipe or by checking to Enable Common Germline Variant Tagging in the BSSH/ICA applications. Note that this flag has implications for other settings. First, annotation must be enabled. Second, if TMB and germline tagging are both enabled, then the TMB database filter needs to be set to false. With these two flags, germline variants are tagged in the VCF but ignored during TMB calculation.

--vc-enable-germline-tagging true
--tmb-skip-db-filter false

If I want both germline and somatic variants, should I run the Tumor sample in the Tumor/Only workflow, because that produces a VCF that contains both germline and somatic variants?

The matched normal provides a more reliable indicator of somatic variants than a database. Database germline tagging for SNV incorrectly tags ~2% of germline variants as somatic and falsely classifies some somatic variants as germline (i.e. false positives and false negatives for somatic SNVs). Additionally, there is limited support for differentiating between germline and somatic CNVs. If a paired normal sample is available, the highest performance for the detection of both somatic and germline variants is achieved by implementing the Tumor/Normal Somatic Variant Calling workflow (as described in this user guide) and also the Germline Caller on the Normal sample.

The workflow is a 2-phase process. Tumor and normal samples need to be run as individual samples for the Map/Align stage, and then Variant Calling is started from tumor and normal UMI collapsed BAM as tumor-normal pairs.

Phase 1: Map/Align

The goal of this phase is to generate UMI collapsed BAM files, and these steps are done using the DRAGEN Enrichment app.

--enable-variant-caller false --umi-start-mask-length 1 --umi-end-mask-length 3 

The Illumina FFPE DNA Prep with Exome 2.5 Enrichment supports the addition of probes during the enrichment process. Spike-in probes are intended to increase coverage in Exome v2.5 regions or add coverage in areas of no coverage by the Exome 2.5 panel. The analysis workflow will be similar, but attention should be paid to the resource files.

Systematic noise file. If the spike-in panel adds coverage to new regions of the genome, it is recommended to generate a new systematic noise file from normal samples enriched with Exome v2.5 plus spike-in. Otherwise, variants calls in the new regions are more vulnerable to false positive calls than regions where, based on the systematic noise file, a somatic filter tackles noise that consistently appears at specific locations in the reference genome.

Panel of Normals (PON) for CNV calls. Whether the spike-in probes add coverage to new regions of the genome or increases coverage of coverage regions, it is always recommended to generate a custom PON, for your specific conditions, if calling CNVs. CNVs are called in regions characterized in the PON, so if the case sample has coverage in additional regions compared to the PON because of a spike-in, analysis will not call CNV events in those additional regions. However, the addition of spike-in probes can change enrichment broadly and may cause coverage fluctuations in previously characterized regions. Thus, CNV calls in regions that are shared by the PON and the case sample could be impacted by the addition of spike-in probes.

You use the DRAGEN Germline Enrichment and the DRAGEN Somatic Enrichment Pipelines in ICA to analyze sequencing data of llumina FFPE DNA Prep with Exome 2.5 Enrichment libraries. The following settings are specific for this library preparation kit.

The workflow is a 2-phase process. Tumor and normal samples need to be run as individual samples for the Map/Align stage, and then Variant Calling is started from tumor and normal UMI collapsed BAM as tumor-normal pairs.

Phase 1 - Preprocessing: Map/Align

The goal of this phase is to generate UMI collapsed BAM files, and these steps are done using the DRAGEN Germline Enrichment app.

Phase 2: Variant Calling

The goal of this phase is perform variant calling, CNV calling, and, if interested, certain biomarkers. The inputs are UMI collapsed BAM from Phase 1.