Wednesday, December 4, 2024
12/4/2024
ABSTRACT Duplex sequencing offers a much-reduced error rate of sequencing and allows for the detection of somatic variation at the single molecule level. Duplex sequencing is part of the proposed experimental design of three GCCs (BCM, Broad and NYGC), although each center plans to use a different technology: NanoSeq/CompDuplex (BCM), CODEC (Broad) or Ultima duplex (NYGC). In addition, TTDs are further developing a tagmentation-based duplex sequencing assay (VISTA- seq, BCH - Choudhury), long-read sequencing with ultralow error rates (HiDEF-seq, NYU) and a single-cell version of the CompDuplex method (BCM). Throughout our existing collaborative project, we have applied each of these technologies to the COLO829 cell line mixture and benchmarking tissues from the SMaHT Network. Following full analysis and dissemination of the benchmarking results, there is a need within the SMaHT Network to i) scale up existing duplex technologies at the GCC, ii) implement duplex technologies developed at TTDs at the GCC, and iii) explore the application of ultradeep, targeted duplex sequencing for variant validation and driver discovery. Here, we seek to address those needs by scaling up production of CODEC at the Broad and by testing, implementing and applying the newly developed HiDEF-seq method. We will apply these methods to all samples from two Tier 1 SMAHT donors. In addition, we will explore using targeted duplex sequencing approaches, based on CODEC, HiDEF-seq or NanoSeq, for variant validation and detection of positive selection through discovery of somatic mutations in key genes or regions.
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