Amplicon library construction and sequencing
For the DNA extractions we used DNeasy PowerSoli kit (Qiagen). 16S rRNA
gene libraries were constructed using primers designed to amplify the
V4 region (Supplementary Table S1) and multiplexed. Amplicons were
generated using a high-fidelity polymerase (Kapa 2G Robust) and purified
using the Agencourt AMPure XP PCR purification system and quantified
using a fluorometer (Qubit, life technologies). The purified amplicons
were then pooled in equimolar concentrations by hand based on Qubit
quantification. The resulting amplicon library pool was diluted to 2 nM
with sodium hydroxide and 5 μl transferred into 995 μl HT1 (Illumina) to
give a final concentration of 10 pM. 600 μl of the diluted library pool
was spiked with 10% PhiX Control v3 and placed on ice before loading
into Illumina MiSeq cartridge following the
manufacturer’s instructions. The sequencing chemistry utilised was MiSeq
Reagent Kit v2 (500 cycles) with run metrics of 250 cycles for each
paired end read using MiSeq Control Software 2.2.0 and RTA 1.17.28. One
of the Pre-adaptation samples (P12) failed in the sequencing run.