Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

Published online 7 April 2016 Nucleic Acids Research, 2016, Vol. 44, No. 11 e105 doi: 10.1093/nar/gkw224 Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing Anders Ståhlberg1,2,* , Paul M. Krzyzanowski3 , Jennifer B. Jackson1 , Matthew Egyud1 , Lincoln Stein3 and Tony E. Godfrey1,* 1 Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA, Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 405 30 Gothenberg, Sweden and 3 Ontario Institute for Cancer Research, MaRS Centre, 661 University Avenue, Suite 510, Toronto, Ontario M5G 0A3, Canada 2 Received November 6, 2015; Revised March 21, 2016; Accepted March 22, 2016 ABSTRACT Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables highlevel multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. INTRODUCTION The ability of massively-parallel, next-generation DNA sequencing (NGS) to identify low prevalence mutations in heterogeneous samples has revolutionized basic and translational research in cancer and many other fields (1). However, detection of sequence variants below 1% frequency remains a challenge with standard NGS protocols due to background noise, much of which is introduced by polymerases during library construction (2). This background noise is problematic in many clinical and research applications, including detection of rare sequence variants in liquid biopsies for non-invasive prenatal diagnostics (NIPD) or for biomarker applications in cancer. Detection and analysis of fetal DNA in maternal plasma has led to a revolution in NIPD for Downs Syndrome and other disorders involving large chromosomal abnormalities (3,4). Moving forward, detection of single nucleotide variants specific to the fetus offers the potential to diagnose monogenic disorders early on in pregnancy without the risks associated with chorionic villus sampling or amniocentesis (5–7). In cancer, applications of rare mutation detection in liquid biopsies include analysis of tumor heterogeneity and identification of therapy resistant clones(8), monitoring clonal evolution and response to therapy (9) and early cancer diagnosis using blood/plasma, sputum, urine or other bodily fluids (10–12). In many cases, these scenarios potentially require detection of variant allele fractions of 0.1% or less. In both NIPD and cancer biomarker research, the introduction of COLD polymerase chain reaction (PCR) (13,14) more recently digital PCR (15) technologies has enabled detection and quantification of ultra-rare sequence variants in liquid biopsies (16,17). However, digital PCR assays are specific for both nucleotide position and the specific base change. Combined with the fact that multiplexing capability is limited, digital PCR is most useful in situations where a known variant is being sought or where disease-related variants are well characterized and limited in number. For * To whom correspondence should be addressed. Tel: +46 31 786 6735; Email: Correspondence may also be addressed to Tony E. Godfrey. Tel: +1 617 414 8013; Email:  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact e105 Nucleic Acids Research, 2016, Vol. 44, No. 11 recessive disorders, mutations in tumor suppressor genes and even recurrent mutations in many oncogenes, de novo detection of variants at many base positions is typically required and digital PCR is not the answer. Instead, sensitive sequencing approaches such as targeted deep sequencing, duplex sequencing or molecular barcoding offer an attractive alternative (18–22) although they typically require complex library construction protocols. Introduction of molecular barcodes (random oligonucleotide sequences e.g. N12-14 ) to uniquely tag individual target DNA molecules can be used to identify and reduce sequencing errors introduced during NGS library construction (Supplementary Figure S1) and enables robust detection of ultra-rare variants (20,23). Ligation of barcodes onto target DNA followed by target capture and amplification is inefficient and risks missing rare variants when using low DNA inputs such as those obtained from liquid biopsies. Introduction of barcodes by PCR can be achieved with low DNA inputs (20) but the random barcode sequences behave promiscuously resulting in formation of non-specific PCR products. Consequently, multiplexing is challenging and library construction requires complex, multi-step workflows, some of which include gel purification of PCR products (20). Here, we report development of a library construction approach that uses reduced primer concentrations, elongated PCR extension times and hairpin-protected barcode primers to enable Simple, Multiplexed, PCR-based barcoding of DNA for Sensitive mutation detection using Sequencing (SiMSen-Seq). SiMSen-Seq facilitates detection of sequence variants at or below 0.1% allele frequency, works with low DNA input (<50 ng) and can be used to interrogate multiple genome loci covering >1 kb of target sequence if desired. MATERIALS AND METHODS DNA Wild-type genomic DNA was extracted from a clonally derived Barrett’s esophageal cell line, CP-A, using the QIAamp DNA Mini kit (Qiagen). Wild-type circulating, cellfree DNA (ccfDNA) was extracted from pooled patien (...truncated)