16S rRNA fecal microbiome cattle fescue toxicosis
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 (Caporaso et al. 2012). DNA sequence data was then generated using Illumina paired-end sequencing at the Environmental Sample Preparation and Sequencing Facility (ESPSF) at Argonne National Laboratory. 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 (Caporaso et al. 2012; Caporaso et al. 2011). The forward amplification primer also contains a twelve base barcode sequence that supports pooling of up to 2,167 different samples in each lane (Caporaso et al. 2012; Caporaso et al. 2011). Each 25 L PCR reaction contained 9.5 L of MO BIO PCR Water (Certified DNA-Free), 12.5 L of QuantaBios 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 then quantified using a fluorometer (Qubit, Invitrogen). After quantification, the molarity of the pool was determined and diluted down to 2 nM, denatured, and then diluted to a final concentration of 6.75 pM with a 10% PhiX spike for sequencing on the Illumina MiSeq.