Genomic DNA was extracted from 0.25 g of fecal material from each sample using the Earth Microbiome DNA extraction protocol28 . Briefly, DNA was extracted using the 96-well format MoBio Powersoil DNA kit on an EpMotion 5075 robot with vacuum (Eppendorf, Hamburg, Germany). DNA was quantified with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
PCR amplification and library preparation were performed similarly to the protocol described by Caporaso et al.29 (link). 515F/806R Illumina primers with unique reverse primer barcodes were used to target the V4 region of the 16S rRNA gene. Samples were amplified in triplicate and cleaned using the MO BIO 69 htp PCR cleanup kit. Each PCR reaction included 1X PCR buffer, 10 μM each forward and reverse primer, 200 μM dNTPs, 1 U/ml Taq polymerase, 15 ng template DNA, and PCR grade water, with a total reaction volume of 25 μL. Reactions were kept at 94°C for 3 minutes for denaturation to occur. Amplification was performed by 25 cycles of 94 °C for 45s, 58 °C for 60s, and 72 °C for 90s. The V4 amplicons were sequenced on the Illumina HiSeq 2000 platform, yielding single end, 100 base pair reads. Sequencing and quality assessment were performed at the Yale Center for Genome Analysis.
PCR amplification and library preparation were performed similarly to the protocol described by Caporaso et al.29 (link). 515F/806R Illumina primers with unique reverse primer barcodes were used to target the V4 region of the 16S rRNA gene. Samples were amplified in triplicate and cleaned using the MO BIO 69 htp PCR cleanup kit. Each PCR reaction included 1X PCR buffer, 10 μM each forward and reverse primer, 200 μM dNTPs, 1 U/ml Taq polymerase, 15 ng template DNA, and PCR grade water, with a total reaction volume of 25 μL. Reactions were kept at 94°C for 3 minutes for denaturation to occur. Amplification was performed by 25 cycles of 94 °C for 45s, 58 °C for 60s, and 72 °C for 90s. The V4 amplicons were sequenced on the Illumina HiSeq 2000 platform, yielding single end, 100 base pair reads. Sequencing and quality assessment were performed at the Yale Center for Genome Analysis.