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Verification of a Software and Amplification-Based Nanopore Sequencing Solution to Characterize Complex Variants in 11 Challenging, High-Frequency Carrier Genes

Prenatal Genetics
  • Primary Categories:
    • Prenatal Genetics
  • Secondary Categories:
    • Prenatal Genetics
Introduction:
Accurate detection of variants associated with severe genetic disorders is crucial for both diagnostic and carrier screening applications. While Next-Generation Sequencing (NGS) is frequently employed, it is often unable to consistently and accurately resolve complex genotypes such as copy number variation (CNV), short tandem repeats, structural variation (SV), and differentiation from highly homologous pseudogenes. As a result, many laboratories must maintain several specialized assays with unique workflows and equipment to identify prevalent complex pathogenic variations, leading to increased costs and operational complexity. We developed a harmonized workflow comprised of a targeted PCR nanopore sequencing assay and accompanying analysis software that examines 11 complex targets (CFTR, SMN1/2, FMR1, HBA1/2, HBB, F8 intron inversions, GBA, CYP21A2, TNXB) representing ~70% of at-risk couples in the US population. Here, we present verification testing performance from studies designed to characterize accuracy, precision, sensitivity, and specificity.

Methods:
Target regions were enriched with 4 PCR mixes in a single assay workflow. Sequencing was performed on a MinION or GridION (Oxford Nanopore Technologies) with R10.4.1 flow cells. Nanopore reads were processed with purpose-built software that provides fully automated quality control, variant reporting, genotype summary, and results visualization tailored to each unique variant type. Verification studies utilized over 425 unique samples that included genomic DNA isolated from whole blood (n >275) with multiple methods (precipitation, column, and magnetic beads) and cell-line genomic DNA (n >150). Verification studies included method comparison, single-site precision, DNA input, analytical specificity, mix modularity, and thermal cycler equivalency. Results were compared to orthogonal data where methods were available.

Results:
The assay demonstrated the ability to identify single nucleotide variants (SNVs), insertions and deletions (indels), CNVs, and SVs with an overall percent agreement (OPA) of >95% in all studies conducted. SNV/indel detection OPA was >98% across all gene targets. SMN1/2 CNV (0 to ≥3) OPA was >95%. FMR1 CGG repeat sizing and AGG interrupt detection OPA was >98%. OPA for HBA1/2 deletion genotypes and CNV and HBB large exon deletions was >95%. Successful differentiation of GBA and CYP21A2 from their respective pseudogenes enabled an OPA of >95% for detecting pathogenic variations. Furthermore, the OPA for detecting large F8 intron inversions was >98%.  The assay was reproducible and robust across operators, runs, DNA extraction methods, mix combinations, and thermal cycler models.

Conclusion:
The harmonized nanopore assay and software system produces precise and accurate results for complex variant classes across 11 targeted genes. The streamlined 2.5-day workflow includes single-day library preparation and automated analysis to identify and report genotypes, visualize data, and predict novel variant impacts to simplify interpretation. By efficiently resolving complex genotypes that cannot be detected by short-read sequencing alone, this technology provides an elegant solution to address the most challenging, yet prevalent genes in carrier screening applications. The streamlined workflow and simplified analysis may increase accessibility and reduce costs for molecular laboratories interested in expanding their complex genotyping capabilities.

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