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Shobha Gokul



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    P3.09 - Pathology (Not CME Accredited Session) (ID 975)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/26/2018, 12:00 - 13:30, Exhibit Hall
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      P3.09-20 - A Simple and Versatile Next-Generation Sequencing Technology for Co-Detection of RNA Structural Variants and DNA Mutations in Lung Cancer (ID 14087)

      12:00 - 13:30  |  Author(s): Shobha Gokul

      • Abstract

      Background

      Variants associated with non-small cell lung cancer (NSCLC) initiation and progression include DNA mutations, copy number variants, RNA fusions and splicing isoforms. Co-detection of DNA and RNA variants has become increasingly important to shorten time from samples to results and optimize personalized medicine for NSCLC. Here we describe the unification of next-generation sequencing (NGS) workflows using library pooling to reliably and sensitively quantify both DNA and RNA variants from tumor FFPE nucleic acids.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      Total nucleic acid (TNA) and DNA were isolated from residual FFPE NSCLC biopsies and cancer cell lines. The QuantideX® NGS RNA Lung Cancer Kit (RUO, Asuragen) and QuantideX® NGS DNA Hotspot 21 Kit (Prototype, Asuragen) were evaluated on the MiSeq® System. DNA and RNA libraries were sequenced on a single MiSeq flow cell. Multiple library pooling strategies were assessed to provide workflow flexibility. All data was analyzed using QuantideX® NGS Reporter 3.0 software.

      4c3880bb027f159e801041b1021e88e8 Result

      The average time from sample QC to MiSeq loading was 10 hours. Including 40-hour instrument run time and data processing and analysis, the sample-to-answer time was less than three days.

      Library pooling experiments evaluated sample run capacity, coverage uniformity, and variant call accuracy from FFPE TNA and DNA. For example, a single pool of 8 RNA and 16 DNA libraries yielded >500,000 reads for each library, with RNA fusions called with 189 to 11,274 reads and DNA mutations detected down to 5% variant allele frequency. Variant calls were 100% concordant with independent results and included mutations in EGFR, RAS, PIK3CA, and BRAF, along with fusions in ALK, RET, and NRG1 and skipped METex14.

      8eea62084ca7e541d918e823422bd82e Conclusion

      The co-detection strategy generated reliable quantitative information from low-input tumor FFPE DNA and TNA within three days. The simplicity and speed of the approach, coupled with a standardized workflow, has the potential to increase the accessibility of NGS analysis and accelerate the return of results for NSCLC patients.

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