Journal: medRxiv

Article Title: Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for Rapid, Accurate, and Affordable Somatic Mutation Detection

doi: 10.1101/2020.11.12.20230169

Figure Lengend Snippet: Validation of the 15-plex OCEANS melanoma panel on melanoma tissue samples. (a) Example results from a fresh-frozen melanoma tissue sample. The BRAF-V600E mutation was the only mutation called for this sample. (b) Example results from a formalin-fixed, paraffin-embedded (FFPE) tissue sample. 5 mutations were called in the AKT1, MAP2K1, MAP2K2, and NRAS genes. Other variants with ≥20% VRF that were not called by Clair are not labeled in the figure. All 5 of these mutations had confirmatory reads on a parallel NGS experiment, but only the NRAS c. 182A>G mutant had a NGS VRF of above 5%; see Supplementary Section S5 for details. (c) Summary of sequencing results for 25 clinical melanoma tissue samples (7 fresh/frozen, 18 FFPE). Input DNA quantities ranged from 10 ng and 50 ng (Supplementary Section S5). The X-axis shows the VRF based on a standard NGS analysis, and the Y-axis shows the OCEANS VRF. The horizontal line shows the 20% VRF cutoff for OCEANS variant calls, and the vertical line shows the 5% VRF cutoff for NGS variant calls. The numbers in quadrants display the number of loci in each group; 97 of the 153 putative variants in the top-left quadrant also had a Clair score of above 180 (purple dots). Relative to the NGS results, the OCEANS panel had a sensitivity of 100% and a specificity of 99.0%. Importantly, we believe that many of the 97 NGS-negative and OCEANS-positive results were true mutations, and our ddPCR confirmation experiments support this hypothesis . We did not observe any significant difference for fresh/frozen samples vs. FFPE samples (Supplementary Section S5). (d) Receiver operator characteristic (ROC) curve for data in panel (c), based on changing the VRF cutoff for OCEANS. The area under the curve (AUC) is 99.99%. (e) High concordance of OCEANS results using Oxford Nanopore MinION vs. Flongle flow cells.

Article Snippet: Comparison experiments against Illumina NGS showed 99.79% to 99.99% area under the receiver-operator curve for these panels on clinical FFPE tumor samples.

Techniques: Mutagenesis, Formalin-fixed Paraffin-Embedded, Labeling, Sequencing, Variant Assay

Journal: medRxiv

Article Title: Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for Rapid, Accurate, and Affordable Somatic Mutation Detection

doi: 10.1101/2020.11.12.20230169

Figure Lengend Snippet: Validation of NSCLC and HCC OCEANS panels on clinical tumor tissue samples. (a) Summary of NSCLC panel results on 23 clinical samples (5 FF, 18 FFPE). Input DNA quantities ranged from 10 ng to 50 ng (Supplementary Section S5). The X-axis shows the VRF based on a standard NGS analysis, and the Y-axis shows the OCEANS VRF. The horizontal line shows the 20% VRF cutoff for OCEANS variant calls, and the vertical line shows the 5% VRF cutoff for NGS variant calls. The numbers in quadrants display the number of loci in each group; 110 of the 136 putative variants in the top-left quadrant also had Clair scores of above 180 (purple dots). 2 of the 11 NGS confirmed variants in the top-right quadrant had Clair scores below 180 (yellow dots); both variants were insertions that typically have lower Clair scores. (b) Receiver operator characteristic (ROC) curve for data in panel (a), based on changing the VRF cutoff for OCEANS. (c) Summary of HCC panel results on 21 clinical samples (5 FF, 16 FFPE). Input DNA quantities ranged from 10 ng to 50 ng (Supplementary Section S5). 118 of the 167 putative variants in the top-left quadrant also had Clair scores of above 180 (purple dots). 3 of the 17 NGS confirmed variants in the top-right quadrant had Clair scores below 180 (yellow dots); all 3 variants were in TERT amplicon within a homopolymer region that resulted in lower Clair scores (Supplementary Section S5). (d) ROC curve for data in panel (c), based on changing the VRF cutoff for OCEANS. (e) Panel (c) showed a significant number of loci wherein NGS results had 0% variant read frequency (VRF). Here, we show a histogram showing NGS read depth distribution for the 0% VRF loci, as well as the fraction of loci for each read depth group where OCEANS VRF was greater than 20%. Only loci with less than 300x NGS read depth showed discordance with OCEANS (mutation call based on 20% VRF).

Article Snippet: Comparison experiments against Illumina NGS showed 99.79% to 99.99% area under the receiver-operator curve for these panels on clinical FFPE tumor samples.

Techniques: Variant Assay, Amplification, Mutagenesis