Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations Austin K. Mattoxa, Yuxuan Wanga, Simeon Springera, Joshua D. Cohena, Srinivasan Yegnasubramanianb, William G. Nelsonb, Kenneth W. Kinzlera, Bert Vogelsteina,c,1, and Nickolas Papadopoulosa,b,d,1 aLudwig Center, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128; bDepartment of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128; cThe Howard Hughes Medical Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128; and dDepartment of Pathology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128 Contributed by Bert Vogelstein, March 13, 2017 (sent for review January 30, 2017; reviewed by Carlos Caldas and Jean-Pierre Issa) The identification of mutations that are present at low frequencies in clinical samples is often far less than optimal, compounding the clinical samples is an essential component of precision medicine. The problem. Sensitivity can be increased by pretreating the DNA to development of molecular barcoding for next-generation sequencing remove damaged bases before sequencing (22, 23) and by bio- has greatly enhanced the sensitivity of detecting such mutations by informatics and statistical methods to enhance base calls after massively parallel sequencing. However, further improvements in sequencing (24, 25). Although useful for a variety of purposes, the specificity would be useful for a variety of applications. We herein sensitivity obtainable with these improvements is generally not describe a technology (BiSeqS) that can increase the specificity of sufficiently high for the most challenging applications, such as sequencing by at least two orders of magnitude over and above that liquid biopsies, which can require detection of one mutant mole- achieved with molecular barcoding and can be applied to any cule among thousands of WT molecules (9). massively parallel sequencing instrument. BiSeqS employs bisulfite Another important way to improve sensitivity is with the use of treatment to distinguish the two strands of molecularly barcoded “molecular barcodes,” in which each template is covalently linked DNA; its specificity arises from the requirement for the same mutation to unique identifying sequences (UIDs). Molecular barcodes were to be identified in both strands. Because no library preparation is originally used to count individual template molecules (26) but required, the technology permits very efficient use of the template were subsequently incorporated into a powerful approach, termed DNA as well as sequence reads, which are nearly all confined to the SafeSeqS, for error reduction (27). After incorporation of the amplicons of interest. Such efficiency is critical for clinical samples, UIDs, subsequent amplification steps produce multiple copies 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 Significance detect one mutation among >10,000 wild-type molecules. The detection of rare mutations in clinical samples is essential next-generation sequencing | bisulfite sequencing | strand-specificity | to the screening, diagnosis, and treatment of cancer. Although polymerase chain reaction | mutation next-generation sequencing has greatly enhanced the sensitivity of detecting mutations, the relatively high error rate of these xtensive knowledge of the genetic alterations that underlie platforms limits their overall clinical utility. The elimination of Ecancer is now available, opening new opportunities for the sequencing artifacts could facilitate the detection of early-stage management of patients (1–3). Some of the most important of cancers and provide improved treatment recommendations tai- these opportunities involve “liquid biopsies”—that is, the evalua- lored to the genetic profile of a tumor. Here, we report the tion of blood and other bodily fluids for mutant DNA template development of BiSeqS, a bisulfite conversion-based sequencing GENETICS approach that allows for the strand-specific detection and quan- molecules that are released from tumor cells into such fluids. Al- tification of rare mutations. We demonstrate that BiSeqS elimi- though the potential value of liquid biopsies was recognized more nates nearly all sequencing artifacts in three common types of than two decades ago (4–6), more recent advances in sequencing mutations and thereby considerably increases the signal-to- technology have made this approach practical. For example, it has noise ratio for diagnostic analyses. recently been shown that liquid biopsies of blood can detect mini- mal amounts of disease in patients with early-stage colorectal Author contributions: A.K.M., S.Y., W.G.N., K.W.K., B.V., and N.P. designed research; A.K.M. cancers, thereby providing evidence that could substantially affect and B.V. performed research; A.K.M., K.W.K., and N.P. contributed new reagents/analytic their survival (7). Other studies have shown that circulating tumor tools; A.K.M., Y.W., S.S., J.D.C., K.W.K., B.V., and N.P. analyzed data; and A.K.M., B.V., and DNA(ctDNA)canbedetectedinthebloodofpatientswithother N.P. wrote the paper. malignancies, as well as in other bodily fluids such as pancreatic Reviewers: C.C., University of Manchester; and J.I., Fels Institute for Cancer and Molecular Biology, Temple University. cysts, Pap smears, and saliva (8–16). Conflict of interest statement: N.P., K.W.K., and B.V. have no conflicts of interest with respect The vast majority of current technologies for detecting rare to the new technology described in this manuscript, as defined by the Johns Hopkins Univer- mutations use digital approaches, where each template molecule is sity policy on conflict of interest. N.P., K.W.K., and B.V. are founders of Personal Genome assessed, one by one, to determine whether it is wild type or Diagnostics, Inc. and PapGene, Inc. K.W.K. and B.V. are members of the Scientific Advisory mutant (17). The digitalization can be performed in wells (17), in Board of Syxmex-Inostics. B.V. is also a member of the Scientific Advisory Boards of Morphotek and Exelixis GP. These companies and others have licensed technologies from Johns Hopkins tiny droplets formed by emulsification or microfluidics (18, 19), or University; N.P., K.W.K., and B.V. are the inventors of some of these technologies and receive in clusters (20). The most comprehensive of these approaches equity or royalties from their licenses. The terms of these arrangements are being managed employs massively parallel sequencing to simultaneously analyze by the Johns Hopkins University in accordance with its conflict of interest policies. the entire sequences of hundreds of millions of individually am- Data deposition: The sequences reported in this paper have been deposited in the Euro- pean Genome-Phenome Archive (EGA) database (accession no. EGAS00001002406; plified template molecules (21). However, all of the currently https://www.ebi.ac.uk/ega/home). available sequencing instruments have relatively high error rates, 1To whom correspondence may be addressed. Email: [email protected] or npapado1@ limiting sensitivity at many nucleotide positions to one mutant jhmi.edu. among 100 wild-type (WT) template molecules, even with DNA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. templates that are of optimal quality (21). The DNA quality of 1073/pnas.1701382114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1701382114 PNAS | May 2, 2017 | vol. 114 | no. 18 | 4733–4738 Downloaded by guest on September 30, 2021 of each UID-linked template. Each of the daughter molecules A produced by amplification contains the same UID, forming a UID family. To be considered a bona fide mutation, termed a super- mutant, every member of the UID family must have the identical sequence at each queried position (27). There are two general ways to assign molecular barcodes to template DNA molecules. One uses a set of locus-specific primers to PCR-amplify genomic loci, while the other uses adapters ligated before amplification to create a whole genome library. The PCR method uses primers containing a stretch of random (N) bases to distinguish each individual template molecule (exogenous barc- B odes) (27, 28). The advantage of this approach is that it is appli- cable to very small amounts of DNA, and virtually the only sequences amplified are the desired ones, reducing the amount of sequencing needed to evaluate a specific mutation. The disad- vantage is that errors introduced into one strand during the UID- incorporation cycles will create supermutants. This method will still therefore eliminate errors during sequencing but not errors made during the initial cycles of PCR. The ligation method either em- – ploys random sequences in the adapters used for ligation (27 29) Fig. 1. Overview of BiSeqS methodology. Bisulfite conversion creates C > T or uses the ends of the randomly sheared template DNA to which transitions at unique positions in each strand. Amplification of the (+)and(–) the adapters are ligated as “endogenous UIDs” (27, 30). Although strands with primers that are amplicon and strand-specific allows for targeted errors are still introduced during the PCR steps with the ligation amplification and addition