title: pooled 16S sequencing: file 6
design: Total DNA from faecal material was extracted using the PowerSoil DNA Isolation Kit (QIAGEN) according to the manufacturers instructions. PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs). The V4 region of the 16S rDNA gene was amplified using the primer set 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACNVGGGTWTCTAAT-3) (where Y = C/T, M = A/C, N = any base, V = A/C/G, W = A/T). These primers were designed with partial Illumina adapters at their 5-end, along with a unique 12 bases barcode for sample identification. The resulting amplicons (PCR1) were visualized using a 1.2% agarose gel. Samples with amplicon sizes ranging from 250 to 500 base pairs (bp) were selected for a second PCR reaction (PCR2). The PCR1 products from ten samples were combined in equimolar ratios to serve as the PCR2 template. PCR2 incorporated a library index sequence and completed the Illumina adapters necessary for sequencing. Subsequently, the amplified PCR2 products were purified, quantified, and sequenced on the HiSeq 2500 platform, generating 250 bp-end reads.