Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands

Authors:

Joshua D Cohen, Christopher Douville, Jonathan C Dudley, Brian J Mog, Maria Popoli, Janine Ptak, Lisa Dobbyn, Natalie Silliman, Joy Schaefer, Jeanne Tie, Peter Gibbs, Cristian Tomasetti, Nickolas Papadopoulos, Kenneth W Kinzler, Bert Vogelstein

Journal:

Nat Biotechnol. 2021 Oct;39(10):1220-1227. doi: 10.1038/s41587-021-00900-z.Epub 2021 May 3.

Abstract:

Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10^-7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.