MiSeq 16S rDNA sequencing and bioinformatic analysis

The DNA concentration of all samples was measured using the Qubit dsDNA High Sensivity Assay Kit (Invitrogen, Carlsbad, CA, USA) with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and diluted to 2 ng/μL prior to PCR amplification. The Bacterial V3-V4 region of 16S rDNA and the Archaeal 16S rDNA were respectively amplified with primers 357F 5′-CCTACGGGNGGCWGCAG-3′ [32] and 805R 5′-GACTACHVGGGTATCTAATCC-3′ [37] and primers 349F 5′-GYGCASCAGKCGMGAAW-3′ and 806R 5′-GGACTACVSGGGTATCTAAT-3′ [34]. Amplicons were generated using a Fluidigm Access Array followed by high-throughput sequencing on an Illumina MiSeq system (Illumina, San Diego, CA, USA) performed at the Carver Biotechnology Center of the University of Illinois (Urbana, IL, USA). The demultiplexed paired end Illumina sequence reads in the FastQ format were uploaded into the Galaxy instance (v.2.3.0) of the Genotoul bioinformatics platform (http://sigenae-workbench.toulouse.inra.fr) to be used in the FROGS (Find Rapidly OTU with Galaxy Solution) pipeline [38]. During the FROGS pre-process, sequences were depleted of barcode and the sequences with a non-appropriate length or containing ambiguous bases were removed. Next, reads were clustered into de novo operational taxonomic units (OTUs) using SWARM algorithm [39] with, at first, a denoising step to build very fine cluster using the minimal distance equal to 1 and, secondly, with an aggregation distance equal to 3. Chimeras were then detected and removed with VSEARCH [40]. Additionally, filters were applied to the OTUs in order to remove singletons [41, 42]. The OTUs selected were taxonomically assigned using the Silva release 132 reference database [43].