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