Rh Caller

The first step of Rh calling is to determine the copy number of RHD and RHCE regions. Reads aligned to the RHD and RHCE regions are counted according to their support of the differentiating sits. The counts in each region are corrected for GC-bias, and then normalized to a diploid baseline. The GC-bias correction and normalization factors are determined from read counts in 3000 preselected 2 kb regions across the genome. These 3000 normalization regions were randomly selected from the portion of the reference genome having stable coverage across population samples.

A collection of 4 differentiating sites in the exon 2 region of RHD and RHCE are used to detect the presence of the RHCE Exon2 gene conversion in the sample. An iterative phasing algorithm is used to build up haplotypes that are supported by the read data. The phasing algorithm starts with candidate haplotypes formed from all possible bases at the first differentiating site. The haplotypes are then extended at the next differentiating site by considering all reads that can be uniquely assigned to a single candidate haplotype. If these reads support only a single base at the next differentiating site for a given candidate haplotype, then the haplotype is extended with that base. When a candidate haplotype can be extended by both bases at the next differentiating site then both possible extended haplotypes are included in the set of candidate haplotypes, growing the set by 1. Subsequent extension steps are performed at neighboring differentiating sites until all sites have been processed. Some haplotypes may have sites that are unresolved (i.e. ambiguous), but these haplotypes can still participate in RHD -> RHCE breakpoint detection.

When the phased haplotypes support the RHCE Exon2 gene conversion. We visit all the differentiating sites ad report them as variants in the output VCF file with ploidy identified using the copy number estimated from the read depth of the differentiating site.

The Rh Caller generates a <output-file-prefix>.targeted.json file in the output directory. The output file is a JSON formatted file containing the fields below.

The rh fields are defined as below.

For the variants the fields are defined as below.

The following are example output files:

{
  "dragenVersion": "4.2.0",
  "sample": "HG002",
  "rh": {
    "totalCopyNumber": 4,
    "rhdCopyNumber": 2,
    "rhceCopyNumber": 2,
    "variants": [
      {
        "hgvs": "NC_000001.11g.25405596_25409676con25283766_25287797",
        "qual": 63.42360372675952,
        "altCopyNumber": 2,
        "altCopyNumberQuality": 74.27989942583051
      }
    ]
  }
}

{
  "dragenVersion": "4.2.0",
  "sample": "HG00110",
  "rh": {
    "totalCopyNumber": 4,
    "rhdCopyNumber": 2,
    "rhceCopyNumber": 2,
    "variants": [
      {
        "hgvs": "NC_000001.11g.25405596_25409676con25283766_25287797",
        "qual": 0.0,
        "altCopyNumber": 0,
        "altCopyNumberQuality": 0.026368888668874203
      }
    ]
  }
}

The Rh Caller also generates a <output-file-prefix>.targeted.vcf[.gz] file in the output directory. The output file is a VCFv4.2 formatted file, possibly compressed.

The following are example output files:

##fileformat=VCFv4.2
...
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT HG002
chr1 25405596 . G A 128.19 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 1/1:128
chr1 25405674 . A T 134.41 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 1/1:134
...
chr1 25409655 . G C 133.16 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 1/1:133
chr1 25409676 . G A 104.46 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 1/1:104

##fileformat=VCFv4.2
...
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT HG00110
chr1 25405596 . G A 44.96 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 0/0:45
chr1 25405674 . A T 35.71 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 0/0:36
...
chr1 25409655 . G C 32.72 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 0/0:33
chr1 25409676 . G A 27.11 PASS EVENT=NC_000001.11g.25405596_25409676con25283766_25287797;EVENTTYPE=GENE_CONVERSION GT:GQ 0/0:27

To enable the Rh Caller, use --enable-rh=true. The Rh Caller is disabled by default. The Rh Caller can run directly from FASTQ input with the mapper or from prealigned BAM/CRAM input. You can also enable the Rh Caller in parallel with any other germline variant callers as part of a WGS germline analysis workflow. For more information on other variant callers, see DNA Pipeline for DRAGEN.

The variants in the <output-file-prefix>.targeted.vcf[.gz] file are included in the <output-file-prefix>.hard-filtered.vcf[.gz] by default. This option can be disabled by removing the rh entry from the --targeted-merge-vc or using --targeted-merge-vc none. The target variants are reported on the RHCE region and connected by the same EVENT in the INFO field.

The output file <output-file-prefix>.targeted.vcf[.gz] is compressed by default. This option can be disabled using --enable-vcf-compression=false.

FASTQ Input

The following command-line example uses FASTQ input:

Copy

dragen \
-r /staging/human/reference/hg38_alt_aware/DRAGEN/8 \
--fastq-file1 /staging/test/data/NA12878_R1.fastq \
--fastq-file2 /staging/test/data/NA12878_R2.fastq \
--output-directory /staging/test/output \
--output-file-prefix NA12878_dragen \
--RGID DRAGEN_RGID \
--RGSM NA12878 \
--enable-map-align=true \
--enable-rh=true

Prealigned BAM Input with VCF merge disabled

The following command-line example uses BAM input that has already been aligned:

Copy

dragen \
-r /staging/human/reference/hg38_alt_aware/DRAGEN/8 \
--bam-input /staging/test/data/NA12878.bam \
--output-directory /staging/test/output \
--output-file-prefix NA12878_dragen \
--enable-map-align=false \
--enable-rh=true \
--targeted-merge-vc none