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.