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

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<sup>-7</sup> 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.

Cohen JD, Dobbyn L, Douville C, Dudley JC, Gibbs P, Kinzler KW, Mog BJ, Papadopoulos N, Popoli M, Ptak J, Schaefer J, Silliman N, Tie J, Tomasetti C, Vogelstein B


Nat Biotechnol, 2021

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