Fig. 1
From: Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis
Two-step PCR scheme used in the PAS method for 16S rRNA gene amplicon sequencing. (a) First-step PCR using regular target gene primers: 515F and 806R to target the v4 region only; (b) Products from the first-step PCR, which do not have the up and down stream sequences of the original template DNA, thus avoiding extra PCR biases resulting from binding of the added components of the long phasing primers to the template DNA; (c) Second-step PCR using complementary phasing primers with spacers of 0–7 bases (generating primer sets of 8 primers, each with a total of 7 bases between the two spacers) in both forward and reverse primers, which provides the sequence position frame shift among samples; (d) Products from the second-step PCR showing the sequence position frame shift among samples