Nextera index kit v2

DNA was extracted using the PowerSoil DNA Isolation Kit (MO-BIO), which has previously been found to be well-suited for fecal samples [35 (link)]. Negative controls were included from DNA extraction [36 (link)]. All plates included a mock community to serve as an internal control for calibration of the bioinformatical pipeline.
The 16S rDNA V3-V4 region was targeted using the primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 [37 (link)]. The following PCR program was used: 98 °C for 30 sec, 25x (98 °C for 10 s, 55 °C for 20 s, 72 °C for 20 s) and 72 °C for 5 min. Amplification was verified by running the products on an agarose gel. Indices were added in a subsequent PCR using the Nextera Index Kit V2 (Illumina), with the following PCR program: 98 °C for 30 sec, 8x (98 °C for 10 s, 55 °C for 20 s, 72 °C for 20 s) and 72 °C for 5 min. The attachment of barcodes was verified by running the products on an agarose gel.
Products from the nested PCR were pooled and the resulting library was cleaned with the Agencourt AMPure XP PCR purification kit (Beckman Coulter). The DNA concentration of pooled libraries was measured on a Qubit fluorometer using the Qubit High Sensitivity Assay Kit (Thermo Fisher Scientific). Sequencing was done on an Illumina MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2x 300 bp paired-end sequencing.

Microbiota composition of fecal samples and mucosal colonic biopsies were analyzed by 16S rDNA sequencing at the Clinical Microbiomics A/S (Copenhagen, Denmark). Briefly, samples were mechanically disrupted using horizontal bead beating on a Vortex-Genie 2 at 2700 rpm for 5 min and total bacterial DNA was isolated using NucleoSpin^® 96 (Macherey-Nagel, GmbH and Co. KG, Düren, Germany). Then, a 16S rDNA Polymerase Chain Reaction (PCR) amplified the V3–V4 region using universal bacterial 16S rDNA primers [37 (link)] with Illumina adapters attached. A second PCR was run and included the Nextera Index Kit V2 (Illumina). The sequencing was carried out on the Illumina MiSeq sequencer using the MiSeq Reagent Kit V3 (Illumina). The analyses of the sequence data were performed using USEARCH (version 10.0) [38 (link)], mothur (version 1.38) [39 (link)], and internal scripts created by Clinical Microbiomics A/S. Reads with 97% sequence identity were clustered as Operational Taxonomic Units (OTUs). The detailed protocol is described in Supplementary Material S2.

The V3–V4 region of the 16S rDNA was amplified using the forward primer
S-D-Bact-0341-b-S-17 (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′) and the reverse primer
S-D-Bact-0785-a-A-21 (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′)⁽⁵⁰⁾, with Illumina adapters attached. The following PCR programme was used: 98°C
for 30 s, 25× (98°C for 10 s, 55°C for 20 s, 72°C for 20 s) and 72°C for 5 min; Nextera
Index Kit V2 (Illumina) indices were added in an identical PCR with only eight cycles.
Products from the PCR reactions were cleaned using the SequalPrep Normalization Plate Kit
(Invitrogen) or Agencourt AMPure XP PCR purification kit (Beckman Coulter) and pooled.
Sequencing was carried out on an Illumina MiSeq sequencer using the MiSeq Reagent Kit V3
(Illumina) for 2× 300-bp paired-end sequencing.