6 months ago by
Walnut Creek, USA
When I first wrote a duplicate-removal program, I was worried about the possible presence of "sister" duplicates, so I designed it to detect both identical and reverse-complementary duplicates. I was not concerned about the presence of these duplicates because DNA is double-stranded and thus a fragment might spawn two clusters - in fact, I doubt that's likely. I assume that when DNA is fragmented, the two strands will generally break independently. Rather, I was concerned that during PCR, a single fragment might spawn both identical and reverse-complementary fragments, so both would need to be removed.
However, in testing, I found that the rate of identical fragments was high, and the rate of reverse-complementary fragments was extremely low - a rate that could be completely accounted for by coincidence. So, I don't worry about it any more. Though I should mention that I don't really understand why this is the case - for a PCR-amplified library it still seems to me that reverse-complementary duplicates should be present. Perhaps someone else has some insight here?