Most third-generation techniques do not require amplification of DNA, which involves using PCR to multiply the DNA many times and therefore can increase the error rate because PCR is unreliable for a large number of replications. Most third-generation techniques can work with a single molecule, so an alternative term for third generation techniques is single-molecule sequencing. Ideally, third-generation sequencing methods also eliminate the need to stop sequencing with a terminator base as in the "wash-and-scan" cycle of second-generation methods, although there are exceptions. Unlike second-generation sequencing, most third-generation sequencing methods rely on the detection of bases while DNA polymerase adds bases, with no need to stop to "scan". Again, as definitions are not completely agreed upon, we will group third generation techniques as techniques which do not require both optical detection and "wash-and-scan" cycle, although some third-generation methods require one or the other. (44)
