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Accurate genotyping of high homology HBA and SMN genes from whole exome sequencing

Laboratory Genetics and Genomics
  • Primary Categories:
    • Genomic Medicine
  • Secondary Categories:
    • Genomic Medicine
Introduction:
Deleterious copy number variants (CNVs) in HBA1/2 and SMN1/2 genes lead to Alpha Thalassemia and Spinal Muscular Atrophy (SMA), respectively. Both diseases are autosomal recessive and have high carrier frequencies, so these two genes are commonly screened to assess carrier status. HBA and SMN genes occur in regions of the genome that are difficult to profile with standard sequencing and bioinformatics workflows due to high homology with paralogous genes. To circumvent these challenges, targeted PCR-based tests (e.g. MLPA) specifically designed to profile HBA and SMN loci are used to supplement NGS panel-based carrier screening assays. These bespoke, gene-specific assays add expense and workflow complexity for laboratories.

Targeted variant callers that accurately genotype HBA and SMN from Illumina whole genome sequencing (WGS) were released in DRAGEN™ v4.2 and v3.9, respectively. Until now, these callers have not handled the normalization of probe capture biases inherent to enrichment-based assays, making whole exome sequencing analysis of those genes infeasible. Further, certain regions used for disambiguation of copy number between paralogous genes are intronic or intergenic and are usually not covered on standard exome panels. Here we present updated versions of the SMN and HBA callers as well as a spike-in panel that can be added to the exome panels.

Methods:
The novel callers leverage the extended coverage of the spike-in probes, panel of normal depth-based normalization, and the specialized CNV detection techniques of the original HBA and SMN callers to enable accurate HBA and SMN genotyping on exome data. This updated solution was tested on a broad set of samples with orthogonally confirmed copy number genotypes for HBA (710 datasets from 203 unique samples) and SMN (728 datasets from 216 unique samples).

Results:
Copy number calls were 100% (710/710) concordant with orthogonal methods for HBA and 97.9% (713/728) concordant with orthogonal methods for SMN1/2. A QC failure rate of about 6% was observed across that sample cohort and a no call rate of 2% for HBA and 1.5% for SMN.

Conclusion:
These data demonstrate the possibility of accurately genotyping HBA and SMN from exome sequencing data by leveraging the newly developed spike-in panel and the novel targeted callers implemented in the DRAGEN™ framework.

 

 

For Research Use Only. Not for use in diagnostic procedures.

 

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