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Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

Significance The detection of rare mutations in clinical samples is essential to the screening, diagnosis, and treatment of cancer. Although next-generation sequencing has greatly enhanced the sensitivity of detecting mutations, the relatively high error rate of these platforms limits their overall clinical utility. The elimination of sequencing artifacts could facilitate the detection of early-stage cancers and provide improved treatment recommendations tailored to the genetic profile of a tumor. Here, we report the development of BiSeqS, a bisulfite conversion-based sequencing approach that allows for the strand-specific detection and quantification of rare mutations. We demonstrate that BiSeqS eliminates nearly all sequencing artifacts in three common types of mutations and thereby considerably increases the signal-to-noise ratio for diagnostic analyses. The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.