Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing

Abstract

Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

Plasma circulating tumor DNA as a potential tool for disease monitoring in head and neck cancer

“…DNA from primary tumor samples and matched normal DNA from buffy coats were sequenced using whole‐exome or targeted next‐generation sequencing panels and mutations were identified using previously published methods . The Mutect2 and Strelka algorithms were used to generate variant call files consisting of several somatic mutations .…”

“…Despite this interest, circulating tumor DNA remains challenging to detect and to quantify as it represents a small fraction of total plasma circulating cell‐free DNA. Novel PCR‐based sequencing approaches have improved our ability to detect rare tumor mutations present in circulating tumor DNA against the total circulating cell‐free DNA background …”

Considerations and quality controls when analyzing cell-free tumor DNA

“…Sequencing libraries were constructed using SiMSen-Seq as described [12]. Briefly, 5 μL of cfDNA was barcoded in a 10 μL reaction, containing 1× Phusion High-Fidelity Buffer, 0.2 U Phusion HF polymerase (both Thermo Fisher Scientific), 0.2 mM dNTP (Sigma-Aldrich), 40 nM of each primer (PAGE-purified, IDT) (Supplementary Table S4), 0.5 M l -carnitine inner salt (Sigma-Aldrich).…”

“…Data were analyzed using Debarcer as described [12,13]. At least three reads with the same barcode (consensus 3) were required to form a valid barcode family.…”

“…Conventional next-generation sequencing (NGS) allows the analysis of multiple target sequences but fails to identify mutations below 1–3% allele frequency. However, NGS methods that correct sequencing errors using barcoding sufficiently improve sensitivity [[10], [11], [12], [13]]. …”