Illumina Connected Analytics (ICA)

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.

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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.

1

Start the DRAGEN Germline Enrichment analysis

Under Pipelines, select either DRAGEN Germline Enrichment 4-3-6 or DRAGEN_Germline_Enrichment_4-3-13. Click Start Analysis on the top right of the page.

2

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.

3

Pricing

Select an Entitlement Bundle from the drop-down menu following Subscription.

4

Input files

Input the fastq (or ORA) files for both the tumor and normal samples.

Although the analysis is implemented in the Germline Enrichment pipeline, both tumor and normal samples are the input data, as the needed output are the UMI-collapsed BAM files.

Reference

The reference genome to use for alignment. Reference genome files are located with the Illumina DRAGEN v10 Reference directory. The provided 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.

Additional Files

Select the same Target BED file selected in Step 6.

5

Settings

Map Align Options

Enable Map/Align—true

Enable Map/Align Output—true

Map/Align Output—bam

Enable Duplicate Marking—false

Variant Calling Options

Enable Small Variant Caller—false

Enable CNV calling—false

Enable SV calling—false

Variant Annotation Options

Enable Variant Annotation—false

Additional Options

Additional DRAGEN Args—Paste the following commands into the box, making sure to update the name of the umi-metrics-interval-file

--enable-umi true -umi-library-type nonrandom-duplex --umi-metrics-interval-file <name_of_file_in_Additional_Files> --umi-min-supporting-reads 1 --umi-start-mask-length 1 --umi-end-mask-length 3 --qc-coverage-ignore-overlaps true

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.

6

Resources

Select the resources settings

7

Start analysis

Once all parameters have been set, click Start analysis on the top right of the page

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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.

1

Start the DRAGEN Somatic Enrichment analysis

Under Pipelines, select either DRAGEN Somatic Enrichment 4-3-6 or DRAGEN_Germline_Enrichment_4-3-13. Click Start Analysis on the top right of the page.

2

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.

3

Pricing

Select an Entitlement Bundle from the drop-down menu following Subscription.

4

Input Files

Input files

Enter the input bam for a tumor-normal pair. Each tumor-normal sample pair requires a different run of the pipeline. Because analysis is being done on a tumor-normal pair, realignment needs to be turned off so both the Tumor and Normal BAM Index (.bai) files need be selected along with the Tumor and Normal BAM files.

Reference

Select the same reference genome used above for alignment.

Target BED file

Select the same target bed file used above for alignment.

Systematic Noise BED File

Select the systematic noise file that matches the Reference genome used for alignment. See the Resource Files for information on pre-built and generating your own file.

CNV Panel of Normals

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 that match the Reference genome selected above and has the desired handling of GC correction. See the Resource Files for information on pre-built target.count files.

5

Settings

General Options

Sample Sex—Choose none from the drop-down list

Map Align Options

Enable Map/Align—false

Enable Map/Align Output—false

Enable Duplicate Marking—false

Variant Calling Options

Enable Small Variant Caller—true

Leave Emit Ref Confidence and VCF File Output blank

Enable CNV calling—true

CNV Use Somatic VC BAF—true

Enable SV calling—false

Targeted Callers

If desired, set Enable Tumor Mutational Burden—true. Note that this requires variant annotation to be enabled.

UMI Options

Enable UMI—false

UMI Library Type—leave blank

UMI Aware Variant Calling—Select Low depth from the drop-down list

Minimum Supporting UMI Reads—remove

Variant Annotation Options

This must be enabled if calculating TMB and adding Germline tagging.

Enable Variant Annotation—true

Variant Annotation Assembly—reference corresponding to the genome used for variant calling

Advanced Options

Additional DRAGEN Args—Paste the following text in the box

--vc-sq-call-threshold 3 --vc-sq-filter-threshold 15

6

Resources

Select the resources settings

7

Start analysis

Once all parameters have been set, click Start analysis on the top right of the page.