16S rRNA library preparation and sequencing

Oligonucleotide primers targeting the V1-V2 hypervariable region of the 16S rRNA gene were selected. PCR was carried out using the following primers,

U28F: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNAGAGTTTGATCMTGGCTCA G-3’

U338R: 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCTGCCTCCCGTAGGAGT-3’

PCR primers were modified version of the standard 28F and 338R primers which contain additional recognition sequences to facilitate nested PCR to add Illumina sequencing adapters and index sequences to resulting amplicons using methods described previously [38].

A second round PCR incorporated Illumina adapters containing indexes (i5 and i7) for sample identification utilising eight forward primers and twelve reverse primers each of which contained a separate barcode allowing up to 96 different combinations. General sequences of the primers are illustrated below with the variable 8 bp barcode underlined and amplification carried out as previously described [38].

N501f 5′AATGATACGGCGACCACCGAGATCTACACTAGATCGCACACTCTTTCCCTACACGACGCT3′

N701r 5′CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTGACTGGAGTTCAGACGTGTGCTC3′.