Risks of double-counting in deep sequencing

Lou et al. (1) report a technique for increased sensitivity of DNA sequencing that they call “circle sequencing.” The authors’ method involves circularizing single-stranded DNA and performing rolling-circle amplification (RCA). Multiple duplicates are thereby generated, and errors can be removed by comparing the sequences of the duplicates. Lou et al. report accuracy similar to that of single-stranded tagging techniques (2, 3), but less than that achieved with double-stranded tagging (4). The authors propose their method as a high-efficiency tool for deep-sequencing of heterogeneous DNA samples. To obtain accurate deep-sequencing from a targeted region, it is necessary to ensure that each starting molecule is …