Using UMIs to reduce amplification bias
One motivation for the use of UMIs is to obtain more accurate relative
abundance data by eliminating possible sequence-specific bias in the PCR
amplification, which may be introduced by variation in polymerase and
primer affinity for some DNA templates. Indeed, we observed that the
Platinum polymerase preferentially amplified the SM170C rpoBallele, whereas the Phusion enzyme did not have this bias (Table
1 and Supplementary Figure S1A-C ). Allele variant bias was
also shown for other target genes, although the ranking of the two
enzymes was not always the same (Table 1 andSupplementary Figures S1-S4 ). However, in our study, the use of
UMIs did not correct the allele bias. This suggests that the bias was
present in the initial round of copying using the target-specific
primer, rather than in the subsequent amplification rounds. For our case
study, at least, the choice of polymerase was much more important for
accurate relative abundance data than the use of UMIs. The main
advantage of UMIs was, rather, the ability to remove most sequencing
errors, as discussed in the preceding section.