BaseSpace Sequence Hub (BSSH) T/N
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. 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 analysis).
Access to Resource Files
The analysis workflow uses resource files. See the Resource Files page for details on where to find those files, which should be manually uploaded to BSSH and available for analyses.
Analysis Settings
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.
Biomarkers
View the Biomarkers options by clicking on + Biomarkers
If HRD calculation is desired, check Enable HRD scoring.
CNV calling must be enabled in order to calculate HRD.
If Tumor Mutational Burden calculation is desired, 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.
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.
Structural Variant (SV) caller results have not been evaluated for accuracy. Calculations for SV can add significant runtime to the analysis.
UMI Settings
These settings will be automatically applied when using the Library Specific Settings.
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.
Variant Annotation must be enabled if calculating TMB.
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.
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