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Oncogene concatenated enriched amplicon nanopore sequencing for rapid, accurate, and affordable somatic mutation detection


Date: 16th November 2020 | Source: medRxiv

Authors: Deepak Thirunavukarasu, Lauren Y. Cheng, Ping Song, Sherry X. Chen, Mitesh J. Borad, Lawrence Kwong, Phillip James, Daniel J. Turner, David Yu Zhang.

Nanopore sequencing is more than 10-fold faster than sequencing-by-synthesis and provides reads that are roughly 100-fold longer. However, nanopore sequencing’s 7.5% intrinsic error rate renders it difficult to call somatic mutations with low variant allele frequencies (VAFs) without significant false positives.

Here, we introduce the Oncogene Concatenated Enriched Amplicon Nanopore Sequencing (OCEANS) method, in which variants with low VAFs are selectively amplified and subsequently concatenated for nanopore sequencing. OCEANS allows accurate detection of somatic mutations with VAF limits of detection between 0.05% and ≤ 1%.

We constructed 4 distinct multi-gene OCEANS panels targeting recurrent mutations in acute myeloid leukemia, melanoma, non-small-cell lung cancer, and hepatocellular carcinoma. Comparison experiments against short-read sequencing showed 99.79% to 99.99% area under the receiver-operator curve for these panels on clinical FFPE tumor samples.

Furthermore, we identified a significant number of mutations below the standard NGS limit of detection in clinical tissue samples using each OCEANS panel. Comparison against digital PCR on 10 of putative mutations at ≤1% VAF showed 9 concordant positive calls with VAFs between 0.02% and 0.66%.

By overcoming the primary challenge of nanopore sequencing on detecting low VAF single nucleotide variant mutations, OCEANS is poised to enable same-day clinical sequencing panels.

Read the full text Cancer research with nanopore Accuracy webpage

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