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

Abstract

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.next-generation sequencing | bisulfite sequencing | strand-specificity | polymerase chain reaction | mutation

Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations

“…Similarly, assymetric chemical labelling of the adapter strands in a way that they can be physically separated can serve an SDE role. A recently described variation of Duplex Sequencing uses bisulfite conversion to transform naturally occurring strand asymmetries in the form of cytosine methylation into sequence differences that distinguish the two strands 86,87 . Although this implementation limits the types of mutations that can be detected, the concept of capitalizing on native asymmetry is noteworthy in the context of emerging sequencing technologies that can directly detect modified nucleotides 88 .…”

Circulating tumor DNA: current challenges for clinical utility

“…To begin to address this vexing issue, recently investigators have explored the use of epigenetic DNA modifications and physical DNA fragmentation patterns to identify not only ctDNA, but which tissue of origin the ctDNA was derived from ( 103 , 104 ). Bisulfite treatment followed by NGS is typically how one can distinguish methylated from unmethylated DNA ( 105 ), but newer approaches using immunoprecipitation have also been published and show high sensitivity and specificity for a given tumor type ( 106 ). These promising approaches set the stage for future clinical utility studies that may realize the full potential of ctDNA and liquid biopsies.…”

Detecting Rare Mutations and DNA Damage with Sequencing-Based Methods

“…Other double-stranded methods include PacBio SMRTbells [35], in which both strands of a DNA fragment are integrated into a circular single-stranded molecule. Also, BiSeqS [42] and muSeq [43] use partial bisulfite treatments to convert a random subset of Cs to Ts to differentiate sense and antisense strands (note that additional information from UMIs or endogenous barcodes would be needed to trace individual reads back to both strands from the same original DNA fragment, which has not yet been implemented in BiSeqS). One limitation of these related approaches is that the C-to-T changes introduced by bisulfite treatment raise obvious challenges for quantifying real C-to-T changes.…”