title: Illumina MiSeq paired end sequencing
design: Goal:  To determine the inherent variability of the gut microbiota between samples obtained from two different areas of the intestinal tract (rectal faeces (RF) and terminal ileum content (TIC) in six identically treated sheep.  Setup of individual library:  Pooled PCR amplicons were sequenced at Edinburgh Genomics, University of Edinburgh, using Illumina miSeq platform. Paired-end sequencing (2x250 bp) was run on the Illumina MiSeq platform. Three separate sequencing primers were used for sequencing; two used to read sequences from either end: Read 1 primer (TATGGTAATT GT GTGCCAGCMGCCGCGGTAA) to yield the 5� read; Read 2 primer (AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT) to yield the 3� read and a third as the indexing primer (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT) used to read the barcode sequence.
library_construction_protocol: Library construction protocol: The bacterial 16S rRNA gene V4 region was amplified for Illumina MiSeq sequencing via a barcoded-adapter based PCR approac. PCR reactions for the bacterial 16S rRNA gene V4 region contained 1x Taq buffer plus additional MgCl2 (final concentration of 1 mM), 0.2 mM of each of the four dNTPs, 0.25 mM of each primer, 0.05 U/?l Taq DNA polymerase (Roche Applied Science), and 1 ng/?l template DNA in a total volume of 25 ?l with PCR grade water. For each amplification reaction the same forward primer (515F) together with a different barcoded reverse primer (806R) were used (the reverse primer sequences differed only at the barcode region). PCR master mixes consisting of nuclease-free PCR grade water, PCR Buffer and MgCl2 were prepared in a sterile PCR cabinet and UV-treated before adding dNTPs, Taq DNA polymerase, forward and reverse primers and template DNA.  Amplification of the bacterial 16S rRNA gene V4 region was as follows:  94 �C for 3 min; followed by 25 cycles at 94 �C for 45 sec, 50 �C for 60 sec, 72 �C for 90 sec; followed by a single cycle of 72 �C for 10 min. For terminal ileal mucosal scraped (TIMS) samples, which contain a low amount of target DNA, 35 cycles of amplification via the above protocol were used. Because of this extended amplification protocol, a PCR negative control showed the presence of amplified sequences corresponding to possible contamination: two such OTUs assigned to g_Staphylococcus and s_Bradyrhizobium elkanii were removed from the dataset as a result.  Size and concentrations of PCR amplicons were analysed by agarose gel electrophoresis. The PCR amplicons were gel-purified using a Wizard� Gel and PCR Clean-Up System (Promega) and quantified using a Quant-iT PicoGreen ds DNA Assay Kit (Life Technologies) before pooling in equimolar quantities for miSeq Illumina sequencing.