HiFi sequencing accurately identifies clinically relevant variants in paralogous genes

Area of Science:

• Genomics
• Genetic Variant Analysis
• Bioinformatics

Background:

• Short-read sequencing (SRS) struggles with genetic variant detection in homologous genomic regions like segmental duplications.
• Paralogous regions with gene-pseudogene pairs often necessitate complex, locus-specific assays for accurate genetic analysis.
• Long-read genome sequencing (lrGS) can span these difficult regions but faces alignment ambiguities for variant calling.

Purpose of the Study:

• To evaluate PacBio HiFi lrGS combined with the Paraphase variant caller for comprehensive variant detection in paralogous genomic regions.
• To assess the capability of lrGS and Paraphase in identifying various genetic variant types, including SNVs, InDels, CNVs, and SVs.
• To determine the readiness of lrGS as a potential first-tier diagnostic approach for variants in challenging genomic loci.

Main Methods:

• PacBio HiFi lrGS was performed on 86 individuals with 125 known variants across 11 paralogous loci.
• Variant calling was conducted using standard HiFi callers and the dedicated haplotype-based caller, Paraphase.
• Performance was evaluated based on the detection rate of known single-nucleotide variants (SNVs), insertions or deletions (InDels), copy-number variants (CNVs), and structural variants (SVs).

Main Results:

• Standard HiFi callers identified 95 out of 125 known variants.
• Paraphase uniquely identified the remaining 30 variants, including SNVs, InDels, CNVs, and SVs.
• Combined, lrGS with Paraphase achieved 100% detection of all known variants and enabled accurate phasing and gene-pseudogene copy-number detection.

Conclusions:

• PacBio HiFi lrGS integrated with Paraphase provides comprehensive variant detection in previously intractable paralogous genomic regions.
• This combined approach significantly enhances the ability to identify diverse genetic variants, including complex structural changes and gene conversions.
• lrGS, particularly with specialized callers like Paraphase, is poised for wider implementation as a diagnostic tool for individuals with suspected variants in challenging genomic regions, pending clinical utility studies.