Browsing by Author "Chen, Sherry X."

Now showing 1 - 3 of 3
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    Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing
    (Springer Nature, 2021) Yan, Yan Helen; Chen, Sherry X.; Cheng, Lauren Y.; Rodriguez, Alyssa Y.; Tang, Rui; Cabrera, Karina; Zhang, David Yu; Bioengineering; Systems, Synthetic, and Physical Biology
    Whole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.
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    Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for rapid, accurate, and affordable somatic mutation detection
    (Springer, 2021) Thirunavukarasu, Deepak; Cheng, Lauren Y.; Song, Ping; Chen, Sherry X.; Borad, Mitesh J.; Kwong, Lawrence; James, Phillip; Turner, Daniel J.; Zhang, David Yu; Bioengineering
    We develop the Oncogene Concatenated Enriched Amplicon Nanopore Sequencing (OCEANS) method, in which variants with low variant allele frequency (VAFs) are 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 construct 4 distinct multi-gene OCEANS panels targeting recurrent mutations in acute myeloid leukemia, melanoma, non-small- cell lung cancer, and hepatocellular carcinoma and validate them on clinical samples. By demonstrating detection of low VAF single nucleotide variant mutations using Nanopore Sequencing, OCEANS is poised to enable same-day clinical sequencing panels.
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    Simultaneous and stoichiometric purification of hundreds of oligonucleotides
    (Springer Nature, 2018) Pinto, Alessandro; Chen, Sherry X.; Zhang, David Yu; Bioengineering
    Purification of oligonucleotides has traditionally relied on mobility-based separation methods. However, these are imperfect, biased, and difficult to scale high multiplex. Here, we present a method for simultaneous purification of many oligonucleotides that also normalizes concentrations. The method uses a rationally designed randomer capture probe to enrich for oligos with perfect 5' sequences, based on the observation that synthesis errors are correlated: product molecules with one or more deletions in one region are also more likely to have deletions in other regions. Next-generation sequencing analysis of 64-plex 70 nt purification products show a median 78% purity, a significant improvement over polyacrylamide gel electrophoresis and high pressure liquid chromatography (60% median purity). Additionally, 89% of the oligo products are within a factor of 2 of the median concentration.