title: Gut microbiota of Behcet's syndrome patients: sample 29B
design: Gut microbiota of a total of 22 Behcet's syndrome patients and 16 healthy co-habiting relatives were characterized by means of barcoded pyrosequencing of the V4 hypervariable regions of the 16S rRNA gene.  Total bacterial DNA from fecal material was extracted using the protocol described in (Salonen et al. J Microbiol Methods, 2010), starting from 250 mg of feces and column-purified.  For the amplification of the V4 region of the 16S rRNA gene the primer set 520F (5’-AYTGGGYDTAAAGNG-3’) and 802R (5’-TACNVGGGTATCTAATCC-3’) (Claesson MJ, et al. PLoS One, 2009) was used. Primers included at their 5’ end an adapter sequence used in the 454-sequencing library preparation protocol, linked to a unique MID tag barcode of 10 bases that allowed the identification of the different samples. Amplifications were carried out as described in (Schnorr SL, Candela M, et al. Nat Comm, 2014).  16S rRNA gene V4 hypervariable region amplicons were individually purified and quantified; then, amplicons were pooled in equal amounts (creating four 6-plex and two 7-plex pools) and purified again in order to remove primer dimers, according to the manufacturer’s instructions.  Purified amplicon pools were quantified using a quantitative Real Time PCR (qPCR) and, afterwards, fixed to microbeads to be clonally amplified by emulsion PCR following the GS-FLX protocol Titanium emPCR LIB-A. Following the amplification step, beads were enriched in order to keep only those carrying identical PCR products on their surface, and loaded onto a picotiter plate for pyrosequencing reactions, following the GS-FLX Titanium sequencing protocol.  Each pool was sequenced in one eighth of a plate.