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Improving coverage of poorly sequenced regions in clinical exomes Eric J. White, Edgardo Lopez, Christopher Gault, Niru Chennagiri, Alexander Frieden, Rebecca Batorsky, Anastasia Nikiforov, Tristen Ross and John F. Thompson Abstract Booster Amplicons Improve Gene Coverage We have previously reported an orthogonal sequencing approach for clinical whole exome sequencing in which results of two next-generation sequencing platforms are combined for rapid variant confirmation. This both reduces the Sanger sequencing confirmation burden by ~95% and increases overall assay sensitivity since each platform uniquely sequences thousands of exons. In the current orthogonal approach, we sequence the Agilent Clinical Research Exome (CRE) libraries on the Illumina NextSeq and combine variants identified from AmpliSeq Exome libraries sequenced on the Ion Torrent Proton. Although the orthogonal platform increases exome variant sensitivity, there are still poorly covered regions that remain and may result in missed pathogenic variants. To minimize this problem, we have designed new sets of primers for low coverage AmpliSeq amplicons and amplified these independently at lower multiplicity than the highly multiplexed standard amplicons. Independent pools are designed for up to hundreds of genes in a phenotypically-driven manner. These are used as a supplement to the standard amplicons and sequenced together with them. We find that many of the low coverage regions are enhanced to the point that variants can be called and sensitivity in those regions is substantially improved so that patients receive a higher quality analysis. The Claritas Clinical Exome forms the backbone of the phenotype specific Region of Interest (ROI) tests offered by Claritas Genomics. While the orthogonal sequencing approach improves coverage and sensitivity relative to either platform alone, small regions of many genes remain that are poorly amplified or sequenced. AmpliSeq primers were designed in a ROI based strategy to increase coverage of genes in the Ion Torrent Proton AmpliSeq Exome sequencing run. Graphs below show cumulative numbers of genes covered at 20X or greater in the individual ROI tests (NA12878). Orange lines show gene coverage of Illumina only. Blue lines show gene coverage of Illumina and Ion Torrent orthogonally combined. Green lines show coverage of orthogonally combined runs with the addition of booster amplicons. Orthogonally Confirmed – 94.4% Proton call matches NextSeq Pass call Reliable 0.3% Proton call matches NextSeq NoPass Likely True Positives Singleton NextSeq call or Singleton 4.6% Proton call with no NextSeq coverage Likely False Positives Singleton NextSeq NoPass or 0.8% Singleton Proton with NextSeq coverage # FP # TP PPV 1 0 49167 99.998% 0 0 134 97.81% 124 ND 2249 94.77% 346 79 ND 18.59% Theaddi=onoftheIonTorrentAmpliSeqExometotheAgilentClinicalResearchExome increasesthenumberoffullycoveredgenesby42%. 20000 18000 16000 14000 Number of Genes Immunology ROI Percent of Gene Covered ≥20x Percent of Gene Covered ≥20x Nephrology ROI Nephrology ROI 12000 10000 Percent of Gene Covered ≥20x Percent of Gene Covered ≥20x Neurology ROI Neurology ROI 6000 4000 Illumina +IonTorrent 2000 0 10 20 30 40 50 60 70 80 90 95 98 99 100 Number of Genes 8000 Number of Genes NumberofGenes Immunology ROI Number of Genes Category Percent of Gene Covered ≥20x Number of Genes % of Total Percent of Gene Covered ≥20x Number of Genes RefSeq intersection NIST intersection CRE intersection AmpliSeq # FPs Fixed by Sanger Bone Marrow Failure ROI Number of Genes Orthogonal sequencing using independent target enrichment and sequencing methods leads to nearly 100% PPV with NA12878 for variants found on both platforms and increases exome assay sensitivity to >98%. Variants found on only one platform are confirmed prior to reporting. 95-100% range Bone Marrow Failure ROI Number of Genes Orthogonal Sequencing Improves Data Quality Full scale PercentofGeneCoveredat≥20x Percent of Gene Covered ≥20x Percent of Gene Covered ≥20x