Comparison of one-step and two-step PCR for 16S Illumina MiSeq. Illumina_PCR_libraryprep_comparison

Despite adoption of high-throughput sequencing of PCR-amplified microbial taxonomic markers for ecological analyses, distinct approaches for preparing PCR amplicon libraries exist. One approach utilises long fusion primers and a single PCR (one-step) while another utilises shorter primers in a first reaction, before transferring diluted amplicons to a second reaction for barcode index incorporation (two-step). We investigated whether transferring diluted amplicons risked creating artificially simplified communities with low biodiversity. In three soils with paired cropland and forest communities, one-step yielded higher alpha-diversity indices, including detection of 2 – 5 times more amplicon sequence variants (ASVs). Modelling expected ASVs per sequence observation predicted that one-step reaches full coverage by 105 sequences per sample while two-step needs 106 – 108. Comparisons of rank abundance demonstrated that two-step covered only 34 – 66% of community distributions. Beta-diversity between cropland and forest centroids was an order of magnitude greater under one-step, indicating greater differences in community composition. Driving differences was underestimation of relatively minor taxa under two-step. These taxa were low in abundance, yet play important roles in carbon cycling, secondary metabolite production, anaerobic metabolism, and bacterial predation. We conclude that one-step amplicon libraries are advisable for studies focussed on diversity or relatively minor yet functionally important taxa.