Changes in mucosal gut microbiota of human volunteers with Coeliac Disease following experimental hookworm infection and consecutive gluten challenges
Genomic DNA was extracted directly from each sample using the Trizol RNA/DNA extraction kit, according to manufacturers’ instructions. High-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed on an Illumina MiSeq platform according to the manufacturers’ protocols with minor adjustments. Briefly, the V3-V4 region was PCR-amplified using universal primers, that contained the Illumina adapter overhang nucleotide sequences, using the NEBNext hot start high-fidelity DNA polymerase (New England Biolabs) and the following thermocycling protocol: 2min at 98°C, 35 cycles of 15s at 98°C - 30s at 63°C - 30s at 72°C, and a final elongation of 5min at 72°C. Amplicons were purified using AMPure XP beads (Beckman Coulter) and the NEBNext hot start high-fidelity DNA polymerase was used for the index PCR with Nextera XT index primers (Illumina) according to the following thermocycling protocol: 30s at 98°C, 8 cycles of 10s at 98°C - 1min 15s at 65°C, and 5min at 65°C. The indexed samples were purified using AMPure XP beads, quantified using the Qubit dsDNA broad range kit (Life Technologies), and equal quantities from each sample were pooled. The resulting pooled library was quantified using the NEBNext library quantification kit (New England Biolabs) and sequenced on an Illumina MiSeq platform using the v3 chemistry (301 bp paired-end reads).
BC2