# Illumina Connected Analytics (ICA) T/N Use the DRAGEN\_Somatic\_Enrichment\_4-3-17 Pipeline as part of the DRAGEN 4.3 Bundle in ICA 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](https://help.connected.illumina.com/illumina-ffpe-dna-prep-with-exome-2.5/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](https://help.connected.illumina.com/illumina-ffpe-dna-prep-with-exome-2.5/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 ICA, 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 ](https://help.connected.illumina.com/illumina-ffpe-dna-prep-with-exome-2.5/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 ](https://help.connected.illumina.com/illumina-ffpe-dna-prep-with-exome-2.5/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 ICA 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

--- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://help.connected.illumina.com/illumina-ffpe-dna-prep-with-exome-2.5/tumor-normal-software-guides/page-2.md?ask= ``` The question should be specific, self-contained, and written in natural language. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.