Microbial 16S rRNA gene amplicon sequencing: mouse stool samples
Briefly, PCR amplicon libraries targeting the 16S rRNA encoding gene                 present in metagenomic DNA were produced using a barcoded primer set adapted for the                 Illumina HiSeq2000 and MiSeq. DNA sequence data were then generated using Illumina                 paired-end sequencing. Specifically, the V4 region of the 16S rRNA gene (515F-806R)                 was PCR-amplified with region-specific primers that include sequencer adapter                 sequences used in the Illumina flowcell. The forward amplification primer also                 contains a twelve-base barcode sequence that supports pooling of up to 2,167                 different samples in each lane. Each 25 ?L PCR reaction contained 9.5 ?L of MO BIO                 PCR Water (Certified DNA-Free), 12.5 ?L of QuantaBio's AccuStart II PCR ToughMix (2x                 concentration, 1x final), 1 ?L Golay barcode tagged Forward Primer (5 ?M                 concentration, 200 pM final), 1 ?L Reverse Primer (5 ?M concentration, 200 pM                 final), and 1 ?L of template DNA. The conditions for PCR were as follows: 94 °C for                 3 minutes to denature the DNA, with 35 cycles at 94 °C for 45 s, 50 °C for 60 s, and                 72 °C for 90 s; with a final extension of 10 min at 72 °C to ensure complete                 amplification. Amplicons were then quantified using PicoGreen (Invitrogen) and a                 plate reader (Infinite? 200 PRO, Tecan). Once quantified, volumes of each of the                 products were pooled into a single tube so that each amplicon was represented in                 equimolar amounts. This pool was then cleaned up using AMPure XP Beads (Beckman                 Coulter) and quantified using a fluorometer (Qubit, Invitrogen). The pool was                 diluted down to 2 nM, denatured, and further diluted to a final concentration of                 6.75 pM with a 10% PhiX spike for sequencing on the Illumina MiSeq. Amplicons were                 sequenced on a 151bp x 12bp x 151bp MiSeq run using previously described sequencing                 primers and procedures. The samples were demultiplexed using                 QIIME.