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

Mutations present at a low frequency in a sample are detected with high sensitivity and a low error rate. 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 x 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.

Automated summary

The SaferSeqS method efficiently detects low-frequency mutations by introducing identical molecular barcodes and enriching target sequences with strand-specific PCR, achieving high sensitivity and specificity, and detecting variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 x 10(-7) mutants per base pair (bp).