To detect somatic copy number aberrations and regions with loss of heterozygosity, run the DRAGEN CNV Caller on a tumor sample with a VCF that contains germline SNVs. The output file is a VCF file. Components of the germline CNV caller are reused in the somatic algorithm with the addition of a somatic modeling component, which estimates tumor purity and ploidy.
The germline SNVs are used to compute B-allele ratios in the tumor, which allows for allele-specific copy number calling on the tumor sample. Where possible, use of the small-variant VCF from a matched normal sample is preferred (tumor-normal mode) for best results, but a catalog of population SNPs can be used when a matched normal sample is not available (tumor-only mode).
When a matched normal sample is available, the sample should first be processed using the germline small variant caller. In this case, only germline-heterozygous SNV sites are used for determining B-allele ratios. If no matched normal is available, population SNP B-allele ratios are computed as for matched normal heterozygous loci, but are treated as variants of unknown germline genotype; possible genotype assignments are statistically integrated to determine allele-specific copy number.
In matched normal mode, a VCF containing germline copy number changes for the individual may optionally be input. This makes sure that germline CNVs are output as separate segments in the somatic whole-genome sequencing (WGS) CNV VCF, and annotated with the germline copy number so that it is clear whether there are specifically-somatic copy number changes in the region.
You can use the following somatic WGS CNV calling command-line options:
The following is an example command line for running tumor-normal somatic WGS CNV calling with a matched normal SNV VCF.
If a matched normal is not available, you must disable CNV calling or run in tumor-only mode. Running with a mismatched normal in tumor-normal mode yields unexpected results. The following example command line runs tumor-only somatic WGS CNV calling with a population SNV VCF.
The following example command line runs tumor normal somatic WGS CNV calling concurrently with the Somatic SNV Caller, which allows you to use the matched normal germline heterozygous sites directly from the SNV Caller with the command cnv-use-somatic-vc-baf true.
You can enable additional features when a matched normal sample and the outputs from DRAGEN Germline analysis are also available. If a matched normal sample is available, enable germline-aware mode and VAF-aware mode using the following example command line. For more information on germline-aware mode and VAF-aware mode, see Germline-aware Mode and VAF-aware Mode.
The target counting stage and its output are the same as for the germline CNV calling case. The target intervals with the read counts are output in a *.target.counts.gz file. If there is insufficient read depth coverage detected, processing will halt. For low depth tumor samples, the value of --cnv-interval-width can be increased from to capture more alignments. The B-allele counting occurs in parallel with the read counting phase, and the values are output in a *.baf.bedgraph.gz file. This file can be loaded into IGV along with other bigwig files generated by DRAGEN for visualization. See Output Files for more details on output files.
The Somatic WGS CNV Caller requires a source of heterozygous SNP sites to measure B-allele counts of the tumor sample. The following are the available modes.
To specify a matched normal sample SNV VCF, use the --cnv-normal-b-allele-vcf option. The VCF file should come from processing the matched normal sample through the DRAGEN germline small variant caller with filters applied. Typically, this file name has a *.hard-filtered.vcf.gz extension. All records marked as PASS that are determined to be heterozygous in the normal sample are used to measure the b-allele counts of the tumor sample. You can also use equivalent gVCF file (*.hard-filtered.gvcf.gz), but the processing time is significantly longer due to the number of records, most of which are not heterozygous sites.
To specify a population SNP VCF, use --cnv-population-b-allele-vcf option. To obtain a population SNP VCF, process an appropriate catalog of population variation, such as from dbSNP, the 1000 genome project, or other large cohort discovery efforts. A suitable example file for this parameter is "1000G_phase1.snps.high_confidence.vcf.gz" from the GATK resource bundle. Only high-frequency SNPs should be included. For example, include SNPs with minor allele population frequency ≥ 10% to limit run time impact and reduce artifacts. Specify the ALT allele frequency by adding AF=<alt frequency> to the INFO section of each record. Additional INFO fields might be present, but DRAGEN only parses and uses the AF field. Sites specified with --cnv-population-b-allele-vcf can be either heterozygous or homozygous in the germline genome from which the tumor genome derives
The following is an example valid population SNP record:
DRAGEN considers the following requirements when parsing records from the b-allele VCF:
Only simple SNV sites.
Records must be marked PASS in the FILTER field.
If there are records with the same CHROM and POS values in the VCF, then DRAGEN uses the first record that occurs.
If a tumor sample and matched normal input are available, use --cnv-use-somatic-vc-baf true. You must enable the Somatic SNV Caller. If using this option, DRAGEN determines the germline heterozygous sites from the matched normal input and measures the b-allele counts of the tumor sample. The information is passed to the Somatic WGS CNV Caller to simplify the overall somatic workflow.
If a tumor sample and matched normal input are available, you can avoid having to separately process the matched normal with the DRAGEN germline pipeline by specifying --cnv-use-somatic-vc-baf true. If using this option, DRAGEN determines the germline heterozygous sites from the matched normal input and measures the b-allele counts of the tumor sample. The information is passed to the Somatic WGS CNV Caller to simplify the overall somatic workflow.
To enable --cnv-use-somatic-vc-baf, enter the following command line options.
--tumor-bam-input <TUMOR_BAM>—Specify the tumor input
--bam-input <NORMAL_BAM>—Specify the matched normal input
--enable-variant-caller true—Enable the somatic SNV variant caller
--cnv-use-somatic-vc-baf true—Enable somatic VC BAF
To specify germline CNVs from a matched normal sample, use --cnv-normal-cnv-vcf. When specified, CNV records marked as PASS in the normal sample are used during tumor-sample segmentation to make sure that confident germline CNV boundaries are also boundaries in the somatic output. Segments with germline copy number changes that are relative to reference ploidy are excluded from somatic model selection. During somatic copy number calling and scoring, the germline copy number is used to modify the expected depth contribution from the normal contamination fraction of the tumor sample. The process leads to more accurate assignment of somatic copy number in regions of germline CNV. DRAGEN then annotates the somatic WGS CNV VCF entries with germline copy number (NCN) and the somatic copy number difference relative to germline (SCND) for the segments that have germline CNVs.
If both the small variant caller and the CNV caller are enabled in a tumor-matched normal run, the somatic SNV results can affect the estimated purity and ploidy of the tumor sample. The somatic SNV variant allele frequencies (VAFs) that are captured by the allele depth values from passing somatic SNVs reflect the combination of tumor purity, total tumor copy number at a somatic SNV locus, and the number of tumor copies bearing the somatic allele. Clusters of somatic SNVs with similar allele depths inform the tumor model.
When a tumor has limited copy number variation and/or CNVs are mostly subclonal, such as in many liquid tumors, VAFs can help prevent incorrect or low-confidence estimated tumor models. Incorrect or low-confidence estimated tumor models can lead to wrong or filtered calls. VAF information can also help determine the presence or absence of a genome duplication even in samples from clonal tumors with clear CNVs.
To utilize VAF information, run somatic WGS CNV calling with small variant calling on tumor and matched-normal read alignment inputs. For example, you could use the following command line:
--enable-variant-caller=true --enable-cnv=true --tumor-bam-input <TUMOR_BAM> --bam-input <NORMAL_BAM>
For tumor/matched-normal runs with --enable-variant-caller true, VAF-based modeling is enabled by default. To disable VAF-based modeling, set --cnv-use-somatic-vc-vaf to false.
DRAGEN uses HET-calling mode for segments with a copy number that is estimated to be heterogeneous (HET) among different subclones. Based on a statistical model, a segment is considered to be heterogeneous when the depths or BAF values in a segment are too far away from what is expected for the closest integer-copy number.
To turn on HET calling, specify --cnv-somatic-enable-het-calling=true on the command line. N.B., this setting will only be honored when DRAGEN is able to identify a confident purity/ploidy model. When a confident model cannot be identified, the caller will return a default model and HET-calling will always be disabled (see Somatic WGS CNV Model section for more details and nuances of this approach).
When a segment is considered as heterogeneous, the output for the segment is changed as follows.
The HET tag is added to the INFO field for the segment.
At least one of the CN and MCN values is given as a non-REF value. Specifically, the values are given as the integer values closest to CNF and MCNF. If the integer values would result in a REF call, then at least one of the CN and MCN values is adjusted to the closest non-REF value.
The ID, ALT, and GT fields are set appropriately for the chosen CN and MCN.
The QUAL score reflects confidence that the segment has nonreference copy number in at least a fraction of the sample.
The CNQ and MCNQ values reflect confidence that the assigned CN and MCN values are true in all of the tumor cells, so at least one of the CNQ and MCNQ values is typically less than five.
Selecting a tumor purity and diploid coverage level (ploidy) is a key component of the somatic WGS CNV caller. The somatic WGS CNV caller uses a grid-search approach that evaluates many candidate models to attempt to fit the observed read counts and b-allele counts across all segments in the tumor sample. A log likelihood score is emitted for each candidate. The log likelihood scores are output in the *.cnv.purity.coverage.models.tsv file. The somatic WGS CNV caller chooses the purity, coverage pair with the highest log likelihood, and then computes several measures of model confidence based on the relative likelihood of the chosen model compared to alternative models.
If the confidence in the chosen model is low, the caller returns the default model with estimated tumor purity set to NA. The default model provides an alternative methodology to identify large somatic alterations (length of at least 1 Mb): records are filtered by this model based on their segment mean value (SM). The threshold values used by the caller are estimated automatically considering the variance of the sample, with larger SM thresholds for DUPs when the variance is higher. The user can use alternative threshold values through the --cnv-filter-del-mean and --cnv-filter-dup-mean parameters. Finally, when the caller returns the default model, the fields regarding copy number states based on model estimation (i.e., CN, CNF, CNQ, MCN, MCNF, MCNQ) are omitted from the final VCF output.
In order to improve accuracy on the tumor ploidy model estimation, the somatic WGS CNV caller estimates whether the chosen model calls homozygous deletions on regions that are likely to reduce the overall fitness of cells, which are therefore deemed to be "essential" and under negative selection. In the current literature, recent efforts tried to map such cell-essential genes¹.
The check on essential regions is controlled with --cnv-somatic-enable-lower-ploidy-limit(default true). Default bedfiles describing the essential regions are provided for hg19, GRCh37, hs37d5, GRCh38, but a custom bedfile can also be provided in input through the --cnv-somatic-essential-genes-bed=<BEDFILE_PATH> parameter. In such case, the feature is automatically enabled. A custom essential regions bedfile needs to have the following format: 4-column, tab-separated, where the first 3 columns identify the coordinates of the essential region (chromosome, 0-based start, excluded end). The fourth column is the region id (string type). For the purpose of the algorithm, currently only the first 3 columns are used. However, the fourth might be helpful to investigate manually which regions drove the decisions on model plausibility made by the caller.
If the somatic WGS CNV caller does not find any overlap between any of the homozygous deletions and any of the essential regions, the model is considered plausible and the model optimization ends. Otherwise, when at least an overlap is found, the model is declared invalid and the model search is repeated on the subset of models that support at least one copy (CN = 1) for the essential region with the lowest coverage among the regions overlapping homozygous deletions.
¹E.g., in 2015 - https://www.science.org/doi/10.1126/science.aac7041
The segmentation stage might produce adjacent or nearby segments that are assigned the same copy number and have similar depth and BAF data. This segmentation can result in a region with consistent true copy number being fragmented into several pieces. The fragmentation might be undesirable for downstream use of copy number estimates. Also, for some uses, it can be preferable to smooth short segments that would be assigned different copy numbers whether due to a true copy number change or an artifact. To reduce undesirable fragmentation, initial segments can be merged during a postcalling segment smoothing step.
After initial calling, segments shorter than the specified value of --cnv-filter-length are deemed negligible. Among the remaining nonnegligible segments, successive pairs are evaluated for merging. On a trial basis, the Somatic WGS CNV Caller combines two successive segments that are within --cnv-merge-distance (default value of 10000 for WGS Somatic CNV) of one another and have the same CN and MCN assignments, along with any intervening negligible segments into a single segment that is recalled and rescored. If the merged segment receives the same CN and MCN as its constituent nonneglible pieces with a sufficiently high-quality score, the original segments are replaced with the merged segment. The merged segment might be further merged with other initial or merged segments to either side. Merging proceeds until all segment pairs that meet the criteria are merged. NB. When the germline CN information is available, and two segments have different germline CN, they will not be merged.
The Somatic WGS CNV Caller can report the total tumor copy number by estimating tumor purity. The BAF estimations from matched normal SNVs or population SNPs allow for allele specific copy number calling. The following table provides examples for a DUP in a reference-diploid region:
*The entry represents a Loss of Heterozygosity (LOH) case. The total copy number is still considered a DUP, so the entry is annotated as GAINLOH to distinguish the value from Copy Neutral LOH (CNLOH), which would be annotated as 2+0.
| Option | Description |
|---|---|
| Option | Description |
|---|---|
| Total Copy Number (CN) | Minor Copy Number (MCN) | ASCN Scenario |
|---|---|---|
--tumor-fastq1,--tumor-fastq2,--tumor-bam-input, --tumor-cram-input
Specify a tumor input file.
--cnv-normal-b-allele-vcf
Specify a matched normal SNV VCF. For more information on specifying b-allele loci, see Specification of B-Allele Loci.
--cnv-population-b-allele-vcf
Specify a population SNP catalog. For more information on specifying b-allele loci, see Specification of B-Allele Loci.
--cnv-use-somatic-vc-baf
If running in tumor-normal mode with the SNV caller enabled, use this option to specify the germline heterozygous sites. For more information on specifying b-allele loci, see Specification of B-Allele Loci.
--sample-sex
If known, specify the sex of the sample. If the sample sex is not specified, the caller attempts to estimate the sample sex from tumor alignments.
--cnv-normal-cnv-vcf
Specify germline CNVs from the matched normal sample. For more information, see Germline-aware Mode.
--cnv-use-somatic-vc-vaf
Use the variant allele frequencies (VAFs) from the somatic SNVs to help select the tumor model for the sample. For more information, see VAF-aware Mode.
--cnv-somatic-enable-het-calling
Enable HET-calling mode for heterogeneous segments. For more information, see HET-Calling Mode.
cnv-normal-b-allele-vcf
Specify a matched normal SNV VCF. Use when a matched normal sample and the matched normal SNV VCF are available. To use this option, you must run the matched normal sample through the DRAGEN Germline workflow.
cnv-population-b-allele-vcf
Specify a population SNP VCF. Use when a matched normal sample is not available and analysis must be performed in tumor-only mode.
cnv-use-somatic-vc-baf
Set to true to enable DRAGEN to identify germline variants during a tumor/matched-normal run, rather than requiring a separate run on the normal sample. Use if and only if tumor and matched normal input are available. Also enable the Somatic SNV Caller via enable-variant-caller to use this option.
4
2
2+2
4
1
3+1
*[LOH]4
0
4+0