Fig. 2

Impact of phasing primers on base frequency distributions within the V4 region of the 16S rRNA gene. The V4 region is typically amplified with the primer set 515F and 806R, which generates an amplicon of 253 bp (excluding primers). Due to high sequence variation, reliable alignments are difficult to obtain for the entire region. Thus, the base frequencies for the first 100 bp from both directions were estimated based on all 16S gene sequences (96,489 for forward direction, 95,071 for reverse direction) from GreenGenes. Differences between the maximum and minimum base frequencies at each sequence position were estimated before and after primer shift for forward (a), and reverse sequences (b). Base frequencies of the first 12 positions of the forward sequences before (c) and after (d) primer shift, and the reverse sequences before (e) and after (f) primer shift