Orthogonal NGS for High Throughput Clinical Diagnostics.

Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

Simultaneous detection of multiple point mutations using allele-specific oligonucleotides.

This unit describes high-throughput mutation analysis using hybridization with pooled allele-specific oligonucleotide (ASO) probes. The approach can be used to screen one gene for many allelic mutations or to screen several loci for several allelic mutations each. Because tetramethyl ammonium chloride (TMAC) is added to the hybridization solution, the melting temperature of each oligonucleotide is independent of G-C content and oligonucleotides of the same length can be hybridized simultaneously. The pooled probes will give a positive hybridization signal from any PCR-amplified DNA sample containing a sequence complementary to any of the ASOs in the pool of oligonucleotide sequences. If many PCR-amplified samples are spotted onto a single membrane, multiple individuals can then be screened simultaneously for many mutant sequences. This multiple ASO hybridization technique is appropriate only for circumstances when hybridization with any one of the pooled probes is expected to be uncommon.