16S rRNA amplicon sequencing
Briefly, microbial cells were lysed using a bead-beater with 0.1-mm glass beads (FastPrep-24 fitted with a cooling adapter). DNA was then purified on NucleoSpin gDNA columns (Machery-Nagel, No. 740230.250). DNA was either used immediately for amplicon analysis or kept frozen as aliquots of 35 ml for metagenomic analysis. After DNA extraction, all pipetting steps until sequencing were conducted using a robotized liquid handler to maximize reproducibility. PCR were conducted in duplicates. DNA was diluted in PCR-grade water and used as template (24ng) for amplifying (25 cycles) the V3-V4 regions of 16S rRNA genes using primers 341F-ovh and 785r-ovh (Kozich et al., 2013) in a two-step process shown to minimize bias (Berry et al., 2011). PCR products were pooled during cleaning using magnetic beads (Beckman Coulter). PCR-fragment concentration was determined by fluorometry and adjusted to 2nM prior to pooling. The multiplexed samples were sequenced on an Illumina HiSeq in paired-end mode (2x250 bp) using the Rapid v2 chemistry. Analysis were based on chimera checked high-quality sequences, samples with low read counts were (< 4,700) were re-sequenced on an Illumina MiSeq using v3