BaseSpace Sequence Hub (BSSH)

PreviousDRAGEN v4.3 Recipe NextIllumina Connected Analytics (ICA)

Last updated 15 days ago

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.

Open the DRAGEN Enrichment App

Version 4.3.6 or newer.

Input Data

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

Configuration

Analysis name—Name of the analysis
Save Result To—Project to store analysis results in.
Input FASTQs—Select the tumor and normal biosample FASTQs.
Sample Sex—Select Unknown.
Variant Caller Mode—Select Germline.

The variant calling mode needs to be specified to launch the application. However, variant calling will not be executed with the Enrichment App and will instead be disabled below by adding an Additional Arguments.

Reference—The reference genome to use for alignment. It is recommended to use linear genome from somatic variant calling, so to use a consistent reference, Homo sapiens [UCSC] hg19 v4 or Homo sapiens [1000 Genomes] hg38 v4 should be used here for mapping.
Targeted Regions—The genomic regions targeted for enrichment. If no spike-in probes were included in enrichment, select Twist Bioscience for Illumina Exome 2.5 Plus Panel from the drop-down list. 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. Refer to .

UMI Settings

View the UMI Settings options by clicking on + UMI Settings
Check the box next to Enable UMI
UMI Library Type—Nonrandom-duplex are used with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment kit
UMI-Aware Variant Calling—select Germline

Even though tumor and normal samples are the input data, Germline is selected in order to be consistent with the Variant Caller Mode selected above. This setting does not impact the UMI collapsed BAM files.

UMI Min Supporting Reads—enter 1. Change from the default of 2.

Advanced Settings

View the Advanced Settings options by clicking on + Advanced Settings
QC Coverage Calculation—Check Do Not Double-Count Overlapping Mates.

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.

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

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

Click Launch Application

Phase 2: Variant Calling

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

Open the DRAGEN Somatic App

Version 4.3.6 or newer.

Input Data

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

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 BAM—Select the tumor BAM, the paired normal BAM, and 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.

Biomarkers

View the Biomarkers options by clicking on + Biomarkers
Biomarkers Tumor Mutational Burden—To calculate TMB, check Enable TMB calculation.

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

UMI Settings

UMI-Aware Variant Calling—Low depth

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.

Click Launch Application

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.

Open the DRAGEN Enrichment App

Version 4.3.6 or newer.

Input Data

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

Configuration

Analysis name—Name of the analysis
Save Result To—Project to store analysis results in.
Input FASTQs—Select the tumor and normal biosample FASTQs.
Sample Sex—Select Unknown.
Variant Caller Mode—Select Germline.

Reference—The reference genome to use for alignment. It is recommended to use linear genome from somatic variant calling, so to use a consistent reference, Homo sapiens [UCSC] hg19 v4 or Homo sapiens [1000 Genomes] hg38 v4 should be used here for mapping.
Targeted Regions—The genomic regions targeted for enrichment. If no spike-in probes were included in enrichment, select Twist Bioscience for Illumina Exome 2.5 Plus Panel from the drop-down list. 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. Refer to .

UMI Settings

View the UMI Settings options by clicking on + UMI Settings
Check the box next to Enable UMI
UMI Library Type—Nonrandom-duplex are used with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment kit
UMI-Aware Variant Calling—select Germline

UMI Min Supporting Reads—enter 1. Change from the default of 2.

Advanced Settings

View the Advanced Settings options by clicking on + Advanced Settings
QC Coverage Calculation—Check Do Not Double-Count Overlapping Mates.

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.

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

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

Click Launch Application

Phase 2: Variant Calling

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

Open the DRAGEN Somatic App

Version 4.3.6 or newer.

Input Data

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

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 BAM—Select the tumor BAM, the paired normal BAM, and 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.

Biomarkers

View the Biomarkers options by clicking on + Biomarkers
Biomarkers Tumor Mutational Burden—To calculate TMB, check Enable TMB calculation.

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

UMI Settings

UMI-Aware Variant Calling—Low depth

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.