# Illumina FFPE DNA Prep with Exome 2.5 Enrichment 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) * Microsatellite Instability (MSI) * Homologous Recombination Deficiency (HRD) * Variant annotation {% hint style="info" %} Structural Variant (SV) caller results have not been evaluated for accuracy. Calculations for SV can add significant runtime to the analysis. {% endhint %} # Demo Data 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 Illumina BioInsight Platform Core 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.

Demo Data in BaseSpace Sequence Hub

# Resource Files ## Distributed as DRAGEN secondary analysis Product Files It is recommended that reference files are based on normal (non-tumorous) samples processed in the same methods used for clinical samples, matching library prep, sequencer instrumentation, etc. When that is not possible, product files to support the analysis of samples prepared with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment are available mostly on the [DRAGEN Resources](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html) page and detailed here. Generally, CNV and MSI resource files show good performance across sequencing instruments, while systematic noise files are highly dependent on the sequence instrument and therefore important to match with clinical samples.
ResourcePrebuiltCustom (Build your own)
Target BEDIf no spike-in probes were included in enrichment, download from Illumina DNA Prep with Exome 2.5 BED files for hg19 or hg38 reference genomes.If spike-in probes were included in enrichment, use a BED file of the combined coverage areas, ie., Exome 2.5 Plus Panel plus the custom panel. The requirements of a BED file are defined here.
Systematic noise files:
considered essential for reducing false positive calls in Tumor-Only workflows, and they are also effective at improving precision in Tumor-Normal workflows.		DRAGEN Resources contains prebuilt systematic noise BED files. The directory SNV Somatic Systematic Noise v2.0.0 contains WES_*_v2.0.0_systematic_noise.snv.bed.gz noise files, which are built from a mixture of FF (fresh-frozen) and FFPE samples, with a mix of TruSeq PCR prep and Nextera prep and sequenced on the NovaSeq 6000. Reference genome builds (the * in the file name) include hg19 and hg38.

What is downloaded from the Resource Page is a tarred directory. Extract the tarball, and within the untarred directory, find the systematic noise file. The g-zipped file can be uploaded to BSSH/Illumina BioInsight Platform Core or used in the command-line execution of DRAGEN.		For instructions on how to build your own systematic noise file using internally-sequenced normal samples, see Custom Systematic Noise Files. Building your own noise file will be necessary if using spike-in probes. The normal samples used to generate the systematic noise file should match as closely as possible the sequencer, sample type, and library prep of the tumor samples. Also available are the DRAGEN Baseline Builder App on BSSH and the Systematic Noise File Builder on Platform Core.
CNV - Somatic pipelines:
A Panel of normals (PONs) is used for calling gene amplification in tumor samples		DRAGEN Resources contains CNV Panel of Normals for Twist Bioscience for Illumina Exome FFPE 2.5 - DRAGEN 4.4 v1.0. The PON was generated from 45 FFPE benign adjacent samples from different tissue types and from male and female donors with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment protocol. Libraries were sequenced on the NovaSeq 6000 sequencing system. Current supported builds include hg19 and hg38, both target.counts and gc-corrected.target.counts.As with the systematic noise file, internally-sequenced normal samples can be used to generate a PON. The input should be the UMI-collapsed BAM files of the normal samples, and the reference genome and target BED need to match those used throughout the workflow. Also available are the DRAGEN Baseline Builder App on BSSH and the DRAGEN CNV Baseline Builder Pipeline on Platform Core.

MSI - Resource directory

To be used when enabling the biomarker MSI in Tumor-only mode.

DRAGEN Resources contains Microsatellite Files v1.1.1. Within the WES directory are *.MSI_baselines_v1.1.0.combined.dist and *microsatellites.list files. The normal samples combined into this baseline files are from benign adjacent samples from different tissue types and from male and female donors. The normal samples were prepared with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment protocol. Libraries were sequenced on the NovaSeq 6000 sequencing system. Current supported builds include hg19 and hg38. The list of microsatellite sites from which to calculate instability are included in the *microsatellites.list files. Note that these microsatellites files do not contain sites on chrY, and that the same genome reference build should be used for the baseline combined dist and microsatellite list files.

What is downloaded from the Resource Page is a tarred directory. Extract the tarball, and within the untarred directory, find the WES directory. Within that directory, are *.combined.dist based on different reference genome builds. Combined .dist files are input for DRAGEN v4.4.		See the DRAGEN manual for instructions on how to generate your own reference directory from interally-sequenced normal samples. Also available are the DRAGEN Baseline Builder App on BSSH and the DRAGEN MSI Baseline Builder 4-3-6 Pipeline on Platform Core.

See the DRAGEN manual for how to generate custom microsatellite lists.
# 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](/additional-information/tumor-only-analysis) 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](https://help.dragen.illumina.com/dragen-v4.4/product-guide/dragen-v4.4/dragen-recipes/dna-somatic-tumor-normal-solid-wes-umi). ```bash /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 # 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](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html) ### Inputs [Here are support pages for the formats of different input file types](https://support-docs.illumina.com/SW/DRAGEN_v39/Content/SW/DRAGEN/Inputfiles_fDG.htm). 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
OptionDescription
--qc-coverage-ignore-overlaps trueresolve 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](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-recipes/dna-somatic-tumor-normal-solid-wes-umi) for tumor-normal analysis is 17.5. ### Annotation Top directory containing Nirvana data file. Instructions on how to download the resource are at . 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 . # BaseSpace Sequence Hub (BSSH) T/N Detailed instructions on how to configure the DRAGEN command-line for analyzing paired tumor-normal samples processed with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment. DRAGEN Somatic App Version 4.4.6 in BaseSpace Sequence Hub is used to analyze sequencing data from llumina FFPE DNA Prep with Exome 2.5 Enrichment **tumor and normal libraries**. Refer to online support for general information about using the [DRAGEN Somatic app](https://www.illumina.com/products/by-type/informatics-products/basespace-sequence-hub/apps/dragen-somatic.html). The settings defined here in this user guide are specific for this library preparation kit and for paired tumor-normal analysis (do not use for [tumor only](/additional-information/tumor-only-analysis) analysis). ## Access to Resource Files The analysis workflow uses resource files. See the [Resource Files](/resource-files) page for details on where to find those files, which should be manually uploaded to BSSH and available for analyses. ## Analysis Settings {% stepper %} {% step %} #### Open the DRAGEN Somatic App Version 4.4.6 {% endstep %} {% step %} #### Input Data Select Biosamples or Samples as the input type to launch the analysis. {% endstep %} {% step %} #### Configuration * Analysis name—Name of the analysis * Save Result To—Project to store analysis results in. * Pipeline Configuration—Select Map/Align + Somatic Small Variant Caller (FASTQ or BAM or CRAM input). * Somatic CNV Calling—Select Tumor-Normal * Input FASTQ—Select the TUMOR FASTQ and the paired NORMAL FASTQ. The Sample Sex can remain as Auto-Detect. 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. Currently *Homo sapiens \[UCSC] hg19 v5* and *Homo sapiens \[1000 Genomes] hg38 v5* are supported for somatic for variant calling. For DRAGEN somatic runs it is recommended to use the linear (non-graph) hashtable. * Library Specific Settings—Select Illumina FFPE DNA Prep with Exome 2.5 Enrichment. Selecting this option presets settings optimized for this sample prep. * Targeted Regions—If no spike-in probes were included in enrichment, select Twist Bioscience for Illumina Exome 2.5 Plus Panel. If the mitochondrial probes were included, select Illumina Exome 2.5 Plus Panel with Mitochondrial Panel. If other spike-in probes were included in enrichment, select Custom BED. 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. The noise file will automatically be selected based on the hg19 or hg38 reference genome specified above. {% endstep %} {% step %} #### Biomarkers * View the Biomarkers options by clicking on + Biomarkers * If HRD calculation is desired, check Enable HRD scoring. {% hint style="info" %} CNV calling must be enabled in order to calculate HRD. {% endhint %} * If Tumor Mutational Burden calculation is desired, check Enable TMB calculation {% hint style="info" %} TMB will be calculated using WES coding regions for the selected genome reference, so Custom TMB Regions do not need to be supplied. {% endhint %} {% endstep %} {% step %} * If Microsatellite Instability calculation is desired, check Enable MSI calling. * MSI References—Select Dataset File(s) and select the combined distance files of the normal reference samples (e.g. WES\_FFPE\_hg\*\_MSI\_baselines\_v1.1.0.combined.dist). The reference genome must be hg19 or hg38. * Custom MSI Regions—Select Dataset File(s) and select the microsatellite list that matches the hg19 or hg38 reference genome used for alignment (e.g. WES\_v1.1.0\_hg\*\_microsatellites.tsv) #### CNV * GC Bias Correction—The correct CNV Combined Counts File will be selected based on the hg19 or hg38 reference genome selected above. {% hint style="info" %} Structural Variant (SV) caller results have not been evaluated for accuracy. Calculations for SV can add significant runtime to the analysis. {% endhint %} #### UMI Settings These settings will be automatically applied when using the Library Specific Settings. {% endstep %} {% step %} #### Advanced Settings * View the Advanced Settings options by clicking on + Advanced Settings * Germline Variant Calling: Uncheck Enable Small Variant Calling on Normal Samples. * Germline Tagging: Uncheck Enable Common Germline Variant Tagging. * Variant Annotation—Check Enable variant annotation. {% hint style="info" %} Variant Annotation must be enabled if calculating TMB. {% endhint %} {% endstep %} {% step %} #### 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\ --qc-coverage-ignore-overlaps true * This specifies that overlapping mates should not be double-counted. {% endstep %} {% step %} #### Click Launch Application {% endstep %} {% endstepper %} # Illumina BioInsight Platform Core T/N Use the DRAGEN\_Somatic\_Enrichment\_4-3-17 Pipeline as part of the DRAGEN 4.3 Bundle in Platform Core to analyze sequencing data of llumina FFPE DNA Prep with Exome 2.5 Enrichment libraries. The settings defined here in this user guide are specific for this library preparation kit and should be used for paired tumor-normal samples. ## Access to Resource Files The analysis workflow uses resource files released with DRAGEN 4.4, so link both the DRAGEN 4.3 and DRAGEN 4.4 Bundles to your project. Additionally, Microsatellite Resource Files need to be manually uploaded and linked to your project if calculating MSI. See the [Resource Files](/resource-files) page for details on where to find those files. Note that the input for the Microsatellite Normal References can be a directory of individual distance files or combined distance file. ## Analysis Settings Start the DRAGEN Somatic Enrichment analysis Under Pipelines, select DRAGEN\_Somatic\_Enrichment\_4-3-17. Click Start Analysis on the top right of the page. ## General User Reference—A run name meaningful to the user User tags, email notifications, and output folders are optional. If no output folder is selected, the output folder will be located in the root of the project. ### Pricing Select an Entitlement Bundle from the drop-down menu following Subscription. ### Input files Input the fastq (or ORA) files for the tumor and normal sample in a sample pair. #### Reference The reference genome to use for alignment. Reference genome files are located with the Illumina DRAGEN v10 Reference directory. The provided [Resource Files](/resource-files) support the use of hg19-alt\_masked.cnv.hla.methylation\_combined.rna\_v4.tar.gz and hg38-alt\_masked.cnv.hla.methylation\_combined.rna\_v4.tar.gz. #### Target BED file BED file that contains targeted regions. If no spike-in probes were included in enrichment, select the Twist\_ILMN\_Exome\_2.5\_Panel bed file that matches the reference genome selected above, stored in the Illumina Enrichment BEDs directory. If other spike-in probes were included in enrichment, generate a BED file of the combined coverage areas (ie., Exome 2.5 Plus Panel with the custom panel), upload to the Platform Core, and select as the target BED file. See the Resources Files for information of format requirements. #### Systematic Noise BED File Select the systematic noise file that matches the panel used for enrichment and the reference genome used for alignment. The Illumina Systematic Noise directory within DRAGEN 4.4 Bundle contains WES\_FFPE\_NovaSeq\_TwistV2.5\_hg\*\_v1.0\_systematic\_noise.bed.gz. See the [Resource Files ](/resource-files)for information on pre-built and generating your own file. #### CNV Panel of Normals/CNV Combined Counts Select the files to comprise the target.counts baseline files. Files must be of the same type (eg, all .target.counts or all .target.counts.gc-corrected.). Select those files that match the panel used for enrichment, match the reference genome used for alignment, and has the desired handling of GC correction. The Illumina DRAGEN CNV Baseline Files/DRAGEN 4.4/\ directory contains combined counts files for both hg19 and hg38. See the [Resource Files ](/resource-files)for information on pre-built target.count files. #### MSI - Microsatellites File and Microsatellites Normal References Directory As noted above, resource files for calculating MSI need to be uploaded to the Platform Core and made available to the Project for inclusion in analysis. #### Microsatellites File Select the microsatellite list that maches the reference genome used for alignment: WES\_v1.1.0\_hg\*\_microsatellites.list #### Microsatellites Normal References Directory/Combined Microsatellites Normal References File Input the directory containing the individual distance files of the normal reference samples (hg\*-WES-FFPE-\*.microsat\_normal.dist), or input a single merged file of the distances for each normal sample. ### Settings #### General Options Output File Prefix—If something other than tumor is desired for the output file prefix, enter that here. Sample Sex—Choose none from the drop-down list #### Map Align Options Enable Map/Align—true Enable Map/Align Output—true Enable Duplicate Marking—false Map/Align Output—bam #### Variant Calling Options Enable Small Variant Caller—true Leave Emit Ref Confidence and VCF File Output blank Enable Germline Tagging—false Enable CNV calling—true CNV Use Somatic VC BAF—true Enable SV calling—false {% hint style="info" %} Structural Variant (SV) caller results have not been evaluated for accuracy. Calculations for SV can add significant runtime to the analysis. {% endhint %} #### Targeted Callers MSI command—tumor-only {% hint style="info" %} It is recommended to run the tumor sample in tumor-only mode for highest performance of this biomarker. Note that this setting allows MSI to be calculated in tumor-only mode while other analyses are calculated in tumor-normal mode. This biomarker is under active development. {% endhint %} MSI Coverage Threshold—60 If desired, set Enable Tumor Mutational Burden—true. Note that this requires variant annotation to be enabled. Enable HLA—false Enable HRD—false {% hint style="info" %} CNV calling must be enabled in order to calculate HRD. {% endhint %} Enable Tumor Mutational Burden—true {% hint style="info" %} TMB will be calculated using WES coding regions for the selected genome reference, so Custom TMB Regions do not need to be supplied. {% endhint %} #### UMI Options Enable UMI—true UMI Library Type—nonrandom-duplex UMI Aware Variant Calling—Low depth Minimum Supporting UMI Reads—1 #### Variant Annotation Options {% hint style="info" %} Variant Annotation must be enabled if calculating TMB. {% endhint %} Enable Variant Annotation—true Variant Annotation Assembly—reference corresponding to the genome used for variant calling #### #### Additional Options Additional DRAGEN Args—Paste the following commands into the box, making sure to update the name of the umi-metrics-interval-file --umi-start-mask-length 1 --umi-end-mask-length 3 --qc-coverage-ignore-overlaps true --vc-sq-call-threshold 3 --vc-sq-filter-threshold 15 {% hint style="info" %} Not Double-Counting Mates provides a more realistic overview of coverage, as reported in the Mean Region Coverage within the Coverage section of DRAGEN Reports. {% endhint %} ### Resources Select the resources setting ### Start analysis Once all parameters have been set, click Start analysis on the top right of the page ## Rerun analysis settings with additional tumor-normal pairs These settings can be rerun on additional tumor-normal sample pairs by selecting the particular analysis and clicking Rerun on the top right corner. Update the User reference and swap out the Tumor and Normal input files for the new pair.

Screenshot highlighting the Rerun feature

# Microsatellite Instability (MSI) Microsatellite Instability (MSI) If MSI values are needed ahead of software updates, it is recommend to calculate MSI on the tumor fastq files in tumor-only mode in a separate DRAGEN analysis. Tumor-o It is recommend to calculate MSI in [tumor-only mode](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-dna-pipeline/biomarkers/biomarker-msi#example-command-for-tumor-only-mode). Tumor-only mode relies on a reference directory of files (.dist) containing the microsatellite repeat distribution in a panel of normals. Both the reference directory and microsatellite list are provided - see the [Resource Files page](/resource-files) for more information - and need to match the reference genome used in the alignment step. # Quick check of run quality Checking coverage on the target regions can be a quick way to determine if the map/align step ran as expected. Following the enrichment and sequencing guidelines, the Mean Target Coverage (MTC) Depth for tumor samples is expected to be ≥130x, for normal samples ≥50x. This metric is called "Average alignment coverage over target region" and can be found in the \.target\_bed\_coverage\_metrics\_\.csv file. Note that results for "Target bed" should be evaluated, as DRAGEN Report defaults to genome, while this is a target-based, WES, assay. Another metric to check is "Aligned reads in target region", which gives a percentage of the reads in the target region relative to the entire genome and can be thought as Percent Read Enrichment.\ \ Other quality metrics to check can include the percentage of Mapped Reads (aim of ≥ 90%) and the Insert length (aim of ≥ 125bp) in the \.mapping\_metrics.csv file. # Spike-in panels 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. **Targeted Regions.** This must be a BED file of the combined coverage areas, ie., Exome 2.5 Plus Panel and the custom panel. Refer to this[ guide on the requirements of the combined BED file](https://help.connected.illumina.com/emedgene/frequently-asked-questions/all-faq/what_is_the_required_format_for_a_bed_file_defining_a_kit). **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. # Germline variants ## Exploring germline variants that might play a role in tumor analysis In somatic T/N analysis, DRAGEN uses a subtraction model for SNVs. VCF will only emit variants where there is no (or very limited) alternate reads in the normal sample. In other words, variants that are found in both the normal and somatic sample will NOT be emitted in the SNV/INDEL VCF (the output \*.hard-filtered.vcf.gz contains only somatic variants). To extract germline variants, it is recommended to run an additional analysis, the[ DRAGEN Germline pipeline](https://help.dragen.illumina.com/product-guides/dragen-v4.3/dragen-recipes/germline-wes), on the normal sample. Note that in this scenario, the germline and somatic variants are output from separate analyses, so the variants may need to be computationally combined. ### Germline variants & Germline tagging in T/N analysis #### Germline variants DRAGEN supports a command-line flag (`--vc-report-germline-variants`, also displayed as "Enable Small Variant Calling on Normal Samples" on the BioInsight platforms) in which the primary output VCF (\*.hard-filtered.vcf.gz) will contain both somatic and germline variants together. However, this setting is **NOT** equivalent to running a dedicated germline caller. The germline calls from T/N somatic mode are a useful approximation for things like LOH analysis and B-allele frequency (used by the Allele-Specific Copy Number (ASCN) module during CNV calling), but they lack the sensitivity tuning that a dedicated germline pipeline provides. #### Germline tagging DRAGEN supports a command-line flag (`--vc-enable-germline-tagging`, also displayed as "Enable Common Germline Variant Tagging" on the BioInsight platforms) that enables post-processing to look up variants in [public databases of human variation](https://help.dragen.illumina.com/dragen-v4.4/product-guide/dragen-v4.4/nirvana) to add INFO/GermlineStatus to the primary output VCF (\*.hard-filtered.vcf.gz). That is, where the VCF in T/N default analysis includes only somatic variants, some of these somatic variants may match a known allele from a database and then be tagged as germline when this flag is enabled. Germline tagging can be seen of as an additional layer of annotation: the matched normal provides one line of evidence (variant not detected in normal → label somatic), while the population database provides another piece of evidence (variant is seen in healthy people → label germline). Note that the germline tagging flag has implications for other settings. First, annotation must be enabled or DRAGEN will error. Second, if calculating TMB is enabled, the additional flag of `--tmb-skip-db-filter` must be set to false or DRAGEN will error. By default in T/N mode, TMB calculation skips the database filter because all variants are somatic in the primary output VCF and consequently included in the TMB calculation. However, if tagging variants as germline, then only those variants labeled as Somatic should be included in the TMB calculation. ### Germline and somatic variants in Tumor-only analysis The matched normal provides a more reliable indicator of somatic variants than comparisons with public database of variants. 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 and additionally the dedicated Germline pipeline on the Normal sample. # Troubleshooting the analysis Potential changes to the analysis settings to improve performance when analyzing Illumina FFPE DNA Prep with Exome 2.5 Enrichment.
Why are the Tumor Mutation Burden (TMB) values not what I expected? **Low TMB values** Variants that do not meet the minimum depth (DP) threshold are excluded from the TMB calculation. The `--vc-callability-tumor-thresh` command line option specifies the threshold value. The default value is 50, and this value assumes at least 100x coverage. If raw coverage is lower than 100x and and tumor purity >= 80%, the threshold value can be decreased to 30, by specifying `--vc-callability-tumor-thresh 30` as an additional argument. **High TMB values** DRAGEN outputs both TMB and NonSyn TMB metrics, where [TMB includes all Filtered Variants in the calculation and NonSyn TMB includes Filtered Nonsynonymous Variants](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-dna-pipeline/biomarkers/biomarker-tmb#id-3.-variant-filters). One metric may be more appropriate than the other for a given comparison. For example, Seracare TMB reference samples include only nonsynonymous variants in their TMB metric.
Why do I have too many/too few PASSing somatic variants? Somatic variants with a quality score less than the threshold value \[0..30] are marked as filtered in the output vcf. The `--vc-sq-filter-threshold`command line option specifies the threshold value. The default value for Tumor/Normal mode is 17.5. We observed that with this library preparation kit, the threshold needed to be lowered slightly to 15 to achieve high sensitivity. Raise this value to improve specificity at the cost of sensitivity, or lower it to improve sensitivity at the cost of specificity.
Why do some qc-coverage metrics report different values between command line and BSSH? There are some rare instances where DRAGEN and BSSH apps have different default settings that might lead to slight difference in coverage calculations. For instance, the BSSH Enrichment App outputs a Aggregate Summary Metrics file that include all reads with MAPQ>=0 in the coverage calculations, while in DRAGEN command-line, MAPQ=0 reads are filtered. To include MAPQ=0 reads in DRAGEN command-line coverage, the flag --qc-coverage-filters-1 “mapq<0” can be added to the recipe.
Why is the CNV VCF file empty? The first thing to check is the coverage of the normal sample. Normal sample counts are added to PON, and if the counts from the normal sample are nearly empty, the GC correction stage will fail. In that case, DRAGEN will output an empty VCF.
# Tumor-only analysis The Illumina FFPE DNA Prep with Exome 2.5 Enrichment kit [supports tumor only analysis](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/ilmn-ffpe-wes-tumor-only-tech-note-m-gl-04165/ilmn-ffpe-wes-tumor-only-tech-note-m-gl-04165.pdf). The DRAGEN v4.4 user guide provides a [recipe for DNA Somatic Tumor-Only Solid WES UMI workflow](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-recipes/dna-somatic-tumor-only-solid-wes-umi), and settings specific for this kit are included here. ``` /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 --validate-pangenome-reference false #currently the linear reference is recommended for somatic analysis # UMI --umi-enable true --umi-library-type nonrandom-duplex --umi-min-supporting-reads 1 --umi-start-mask-length 1 --umi-end-mask-length 3 # 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-enable-germline-tagging true --vc-enable-non-primary-allelic-filter true --vc-enable-triallelic-filter false # CNV --enable-cnv true --cnv-population-b-allele-vcf $POP_SNPs #Path to population SNP VCF; see https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-dna-pipeline/cnv-calling/additional-documentation/cnv-preprocessing#b-allele-counts-ascn-callers --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 --tmb-enable-proxi-filter 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 --msi-microsatellites-file ${microsatellite_file} #see Resource Files page --msi-ref-normal-dir ${normal_reference_directory} #see Resource Files page ```