SMN Caller

Two common copy-number variants (CNVs) in SMN1 and SMN2 include whole gene CNV and a partial gene deletion of exons 7 and 8.

Reads that align to either SMN1 or SMN2 are counted. The read counts in exon 1 through exon 6 are used to determine total SMN copy number. The read counts in exon 7 and 8 are used to determine the SMN copies that do not have the exon 7 and 8 deletion (intact SMN copy number). To estimate the SMN copy number for these two regions, read counts are normalized to a diploid baseline derived from 3000 preselected 2 kb regions across the genome. The 3000 normalization regions are randomly selected from the portion of the reference genome that has stable coverage across population samples. The SMN Caller then calculates the number of SMN copies that have the exon 7 and 8 deletion by subtracting the intact SMN copy number from the total SMN copy number.

To calculate the SMN1 copy number, the caller uses eight predefined differentiating sites in exons 7 and 8 of SMN1 and SMN2. One of these sites is the splice site variant used for SMA calling with ExpansionHunter (see SMA Calling With ExpansionHunter). The caller selects differentiating sites at positions that have sequence differences between SMN1 and SMN2 where calling the SMN1 copy number is most likely to be correct based on sequencing data from the 1000 Genomes Project.

For each differentiating site, the SMN1-specific and SMN2-specific alleles are counted in reads mapping to either SMN1 or the homologous region in SMN2. The caller uses a binomial model to calculate the likelihood of each possible SMN1 copy number from the two gene-specific counts given the intact SMN copy number calculated in the previous step.

The SNP c.*3+80T>G (also referred to as g.27134T>G in literature) is associated with “silent” carriers of SMA when the copy number of SMN1 is 2 i.e., both copies of the SMN1 gene are located on the same haplotype and the other haplotype has a deletion of the gene. This variant is located on intron 7 of SMN1 and its copy number is computed using the same strategy as used for calling the copy number at differntiating sites.

The SMN Caller prints out its calls in the targeted callers output file, <prefix>.targeted.json (that also aggregates calls from other targeted callers). An example of this file with the SMN caller set is as follows:

{
    "dragenVersion": "4.2.0-724-gb600fcef",
    "sample": "NA19374",
    "smn": {
        "smn1CopyNumber": 3,
        "smn2CopyNumber": 1,
        "smn2Delta78CopyNumber": 0,
        "totalCopyNumber": "3.89",
        "fullLengthCopyNumber": "4.10",
        "variants": [
        {
            "hgvs": "NM_000344.4:c.*3+80T>G",
            "qual": null,
            "altCopyNumber": 1,
            "altCopyNumberQuality": 54.63854344932561
        }
        ]
    }
}

For SMN caller, the fields are defined as follows.

The variant NM_000344.4:c.*3+80T>G is also reported in a <output-file-prefix>.targeted.vcf[.gz] file in the output directory. The output file is a VCFv4.2 formatted file and possibly compressed. The variant is reported with the VARIANT_IN_HOMOLOGY_REGION flag in the INFO field and also filtered with the TargetedRepeatConflict filter. This variant lies in a region of homology between SMN1 and SMN2 and hence this variant is reported twice - once for each SMN1 and SMN2 regions - and is connected by the same EVENT in the INFO field. The ploidy of the variant is reported in concordance with the identified genotype.

An example of the vcf entry for the variant NM_000344.4:c.*3+80T>G is as follows.

##fileformat=VCFv4.2
...
#CHROM    POS    ID    REF    ALT    QUAL    FILTER    INFO    FORMAT    HG04038
chr5    70076654    .    T    G    150    TargetedRepeatConflict    EVENT=NM_000344.4:c.*3+80T>G;EVENTTYPE=VARIANT_IN_HOMOLOGY_REGION    GT:GQ    0/0/0/1:55
chr5    70952074    .    T    G    150    TargetedRepeatConflict    EVENT=NM_000344.4:c.*3+80T>G;EVENTTYPE=VARIANT_IN_HOMOLOGY_REGION    GT:GQ    0/0/0/1:55
...

The variant NM_000344.4:c.*3+80T>G in the <output-file-prefix>.targeted.vcf[.gz] file can also be included in the <output-file-prefix>.hard-filtered.vcf[.gz] by including the smn entry int the --targeted-merge-vc list, i.e. --targeted-merge-vc smn. The output file <output-file-prefix>.targeted.vcf[.gz] is compressed by default. This option can be disabled using --enable-vcf-compression=false.

Setting the option --targeted-enable-legacy-output=true the caller also generates an SMN specific output file called <output-file-prefix>.smn.tsv in the output directory, which is deprecated for DRAGEN v4.2. The output file contains a header line followed by a sample call line that contains the following tab-delimited fields.

* The value None indicates a confident call could not be made.

The following is an example <output-file-prefix>.smn.tsv output file.

#Sample  isSMA  isCarrier  SMN1_CN  SMN2_CN  SMN2delta7-8_CN  Total_CN_raw  Full_length_CN_raw  SMN1_CN_raw
HG00111  False  False      2        3        0                4.88          5.04                1.00,2.74,2.10,1.89,2.21,1.63,2.47,2.00

To enable the SMN Caller, use --enable-smn=true or --enable-targeted=true (the option --enable-targeted=true also enables other DRAGEN targeted callers). The SMN Caller is disabled by default. The SMN Caller can run from FASTQ input with the mapper enabled or from prealigned BAM/CRAM input. You can also enable the SMN Caller in parallel with any other germline variant callers for a WGS germline analysis workflow. For information on other variant callers, see DRAGEN DNA Pipeline.

FASTQ Input

The following command-line example uses FASTQ input.

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-smn=true

Prealigned BAM Input

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

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-smn=true