Browsing by Author "Cheng, Lauren Y."

Now showing 1 - 4 of 4
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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; 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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    Direct capture and sequencing reveal ultra-short single-stranded DNA in biofluids
    (Cell Press, 2022) Cheng, Lauren Y.; Dai, Peng; Wu, Lucia R.; Patel, Abhijit A.; Zhang, David Yu
    resulting fluorescence images and compared with tissue histopathology maps. The EGFﾖAlexa 647 signal correlated well with EGFR expression as indicated by immunohistochemistry. A classification algorithm for presence of neoplasia based on the signal from both contrast agents resulted in an area under the curve of 0.83. Regions
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    High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens
    (Springer Nature, 2021) Cheng, Lauren Y.; Haydu, Lauren E.; Song, Ping; Nie, Jianyi; Tetzlaff, Michael T.; Kwong, Lawrence N.; Gershenwald, Jeffrey E.; Davies, Michael A.; Zhang, David Yu; Systems, Synthetic, and Physical Biology
    Mutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.
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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
    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.