Bacterial 16S rRNA Profiling by Illumina Sequencing

Frozen pellet obtained from 20 ml sample extract after stomaching were centrifuged and DNA extraction was performed on the pellet. Nucleospin® tissue kit (Macherey-Nagel) was used for the extraction according to manufacturer instructions. Concentration and quality of extracted DNA were checked using a BioSpec-nano spectrophotometer (Shimadzu). The bacterial V3-V4 region of the 16S rRNA gene was then amplified using the forward primer 5′-CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCAGCAG-3′ and the reverse primer 5′-GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGGGTATCTAATCCT-3′^{23 (link)}. The preparation of amplicons was performed in a total volume of 50 µL containing 0.5 µl of TAQ Polymerase (5 U/µl) and 5 µl of adequate 10 X PCR buffer (MTP Taq DNA Polymerase, Sigma), 1 µl of 10 mM dNTP (Sigma), 1.25 µM of each primer (20 µM) and 10 ng of DNA template. PCR was performed using a LightCycler 480 thermocycler as follows: 94 °C for 60 s for initial melting; 30 cycles at 94°c for 60 s, 65 °C for 60 s, 72 °C for 60 s; and 72 °C for 10 min following by incubation at 4 °C. Products were then verified on a 1.5% agarose gel before being sent to GeT-PLaGe Genotoul plateform (Castanet-Tolosan, France) for paired-end sequencing on Illumina MiSeq platform (Illumina, USA) at a read length of 2 × 250 pb.