This recipe is for processing whole genome sequencing data for somatic heme tumor only workflows.
For most scenarios, simply creating the union of the command line options from the single caller scenarios will work.
Configure the INPUT options
Configure the OUTPUT options
Configure MAP/ALIGN depending on if realignment is desired or not
Configure the VARIANT CALLERs based on the application
Configure any additional options
Build up the necessary options for each component separately, so that they can be re-used in the final command line.
The following are partial templates that can be used as starting points. Adjust them accordingly for your specific use case.
Optional settings per component are listed below. Full option list at this page.
Generic SNV noise files (including a HEME specific WGS noise file) can be downloaded here: DRAGEN Software Support Site page
When possible it is recommended to build a pipeline specific systematic noise file that matches the library prep and sequencer of interest:
Step 1. Run DRAGEN somatic tumor-only on each of approximately 50 normal samples:
Gather the full paths to the VCFs from step 1 in ${VCF_LIST} by specifying 1 file per line.
Step 2. Generate the final noise file with:
To build the SV systematic noise file
You can generate systematic noise BEDPE files from normal samples collected using library prep, sequencing system, and panels.
To generate a BEDPE file, do as follows.
Run DRAGEN somatic tumor-only on normal samples with --sv-detect-systematic-noise set to true to generate VCF output per normal sample.
Build the BEDPE file using the VCFs and the --sv-build-systematic-noise-vcfs-list: List of input VCFs from previous step. Enter one VCF per line. Example command line is provided below
You can also build systematic noise BEDPE files in the cloud using the DRAGEN Baseline Builder App on BaseSpace.
Pre-built SV systematic noise file
The following prebuilt systematic noise files for WGS are available for download on the DRAGEN Software Support Site page. To generate these noise files, we used 46 unrelated normal samples.
We recommend using --enable-variant-deduplication true to filter all small indels in the structural variant VCF that appear and are passing in the small variant VCF (PASS in the FILTER column of the small variant VCF file). Using this feature, DRAGEN will create a new VCF that contains variants in SV VCF that are not matching a variant from SNV VCF file. The new deduplicated SV VCF file will have the same prefix passed by --output-file-prefix followed by sv.small_indel_dedup.vcf.gz. DRAGEN normalizes variants by trimming and left shifting by up to 500 bases. An instance of utilizing this feature is when incorporating both SV and SNV callers in somatic workflows, which can increase sensitivity and prevent the occurrence of replicated variants within genes such as FLT3 and KMT2A.
| Option | Description |
|---|---|
| Option | Description |
|---|---|
| Option | Description |
|---|---|
| Pre-built Systematic Noise File | Comment | Systematic Noise Version | DRAGEN Compatibilit |
|---|---|---|---|
--heme-cnv true
Configures DRAGEN to use CNV settings for Liquid Tumors (e.g., AML/MLL).
--vc-sq-filter-threshold $THRESHOLD
Threshold for sensitivity-specificity tradeoff. The default threshold is 3. Raise this value to improve specificity at the cost of sensitivity, or lower it to improve sensitivity at the cost of specificity.
--vc-systematic-noise $SYSTEMATIC_NOISE_FILE
Systematic noise filter. In tumor-only variant calling, this filter is essential for removing systematic noise observed in normal samples. Prebuilt systematic noise files are available for download on the DRAGEN Software Support Site page. Alternatively, a systematic noise file can be generated by running the somatic TO pipeline on normal samples. We recommend using a systematic noise file based on normal samples that match the library prep of the tumor samples.
--vc-somatic-hotspots somatic_hotspots_GRCh38.vcf.gz
Hotspots file. By default, DRAGEN treats positions in the COSMIC database as hotspots, assigning an increased prior probability to variants at these positions. Use this option to override a custom hotspot file if a list of positions of interest is available.
--vc-combine-phased-variants-distance $DIST
Combining phased variants. By default, DRAGEN will not combine nearby phased calls into MNVs or indels. To combine such calls, set this parameter to a value greater than zero indicating the maximum distance at which calls should be combined. If the user wants to enable the combining of phased variants the recommended value of the distance is 15 base pairs. The valid range is [0; 15]
--vc-enable-germline-tagging true --enable-variant-annotation true --variant-annotation-data $NIRVANA_ANNOTATION_FOLDER --variant-annotation-assembly $REFERENCE
Germline filtering. Enable to tag variants as germline or somatic based on population databases. $REFERENCE can be GRCh37 or GRCh38 (GRCh37 is compatible with hs37d5 and hg19). The Nirvana annotation database is downloadable at this page.
--vc-target-vaf FLOAT
This option is only available starting in V4.2. The vc-target-vaf is used to select the variant allele frequencies of interest. The variant caller will aim to detect variants with allele frequencies equal to and larger than this setting. This setting will not apply a hard filter and it is possible to detect variants with allele frequencies lower than the selected threshold. On high coverage and clean datasets, a lower target-vaf may help increase sensitivity. On noisy samples (like FFPE) a higher target-vaf maybe help reduce false positives. Using a low target-vaf may also increase runtime. The valid range is [0, 1]. The default is 0.03 (or 0.001 when --vc-enable-umi-liquid=true).
--sv-systematic-noise $SV_SYSTEMATIC_NOISE_BEDPE
Systematic noise BEDPE file containing the set of noisy paired regions (optionally gzip or bzip compressed). For more information, see Systematic Noise Filtering.
--heme-sv true
configures DRAGEN to use SV settings for Liquid Tumors (e.g., AML/MLL).
--sv-min-scored-variant-size $MIN_SCORED_VAR_SIZE
100000
--sv-somatic-ins-tandup-hotspot-regions-bed $BED_FILE
Specify a BED of ITD hotspot regions to increase sensitivity for calling ITDs in somatic variant analysis. By default, DRAGEN SV automatically selects a reference-specific hotspots BED file. The default file includes FLT3, ARHGEF7, KMT2A, and UBTF exonic regions with some padding on both sides (300 bps)
IDPF_WGS_hg38_v3.0.0_systematic_noise.sv.bedpe.gz
>200x coverage with 2x150bp reads for the HG38 reference
3.0.0
4.3.*