5 sequencing adapters

For identification of bacterial communities, primers specific to the variable region V3 to V4 of the 16S rDNA of bacterial species were used, the forward primer was 341F and the reverse primer was 806R. Besides, the primers also contained Illumina 5′ sequencing adapters, so that the resultant V3-V4 amplicons were incorporated with Illumina 5′ sequencing adapters. Details of the primers are shown in Supplementary Table S1. Amplification was performed using 10 μl 2× Phusion High-Fidelity PCR Master Mix (New England Biolabs, Beverly, MA, United States), 0.4 μM each primer, and 5 μl of genomic DNA. PCR conditions involved a denaturation at 98°C for 30 s, 30 cycles of denaturation at 98°C for 15 s, annealing at 60°C for 15 s, elongation at 72°C for 15 s, and final extension at 72°C for 10 min.
Suitability of the V3 and V4 primers was evaluated in silico using the online tool arb-SILVA TestPrime (Quast et al., 2012 (link)) based on the 16S small subunit (ssur123) and non-redundant SILVA reference database (SILVA Ref NR). At one-mismatch-stringency, the V3-V4 specific primer pairs (excluding the Illumina 5′ sequencing adapters) showed 87% coverage for all bacterial phyla and considered suitable.

The V3-V4 PCR products generated were 1/20-diluted, 1 μl of the diluted amplicon was used in the second PCR step. The second PCR incorporated Illumina flow-cell linkers and 8-bp dual-index barcodes to the amplicons. This was done using primers with Illumina flow cell linkers + 8-bp index barcodes + Illumina 5′ sequencing adapters. Primer sequences are shown in Supplementary Table S1. PCR was performed using 10 μl 2× Phusion High-Fidelity PCR Master Mix (New England Biolabs), 0.4 μM of each primer and 1 μl of the diluted amplicon. PCR conditions involved an initial denaturation at 98°C for 30 s, 10 cycles consisting of denaturation at 98°C for 15 s, annealing at 60°C for 15 s, elongation at 72°C for 15 s, and final extension at 72°C for 10 min.
After PCR amplification, amplicons were pooled in equal volume, the amplicon library was purified using QIAgen PCR Purification kit (QIAgen, Hilden, Germany). Quality of the library was assessed on Agilent Bioanalyser 2100 system (Agilent Technologies Inc., Santa Clara, CA, United States). The library was sequenced on an Illumina HiSeq2500 system (Illumina, San Diego, CA, United States) and 250 bp paired-end reads were generated.