title: Illumina MiSeq paired end sequencing
design: Goal:  To define the effect of infection with, and treatment of, sensitive and resistant strains of T.circumcincta on the ovine terminal ileum mucosal scraped (TIMS) microbiota and rectal faecal (RF) composition.
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.