Genome sequencer gs flx titanium platform

PCR amplification, purification, pooling, and pyrosequencing were performed following the procedure described by Liu et al. [15 ], with minor modifications. The V1–V3 hyper-variable region of the bacterial 16S rDNA was amplified with primers B-27F and A-533R (454 Life Sciences, Branford, CT, USA). Sequencing starts from A-533R to B-27F (A and B represent the 454 adaptors, where A contains the index sequences). PCR was carried out in triplicate 20-μL reactions containing 100 nM of each primer, 10 ng of template, 0.25 mM dNTPs, and 1 U polymerase (TransStart-FastPfu DNA Polymerase, TransGen Biotech, Beijing, China). The following thermal program was used for amplification: 95°C for 2 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and then an extension at 72°C for 5 min. The PCR products were pooled and purified. Equal amounts of the products from each sample were combined in a single tube to be run from the A-end on a Roche Genome Sequencer GS-FLX Titanium platform at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

DNA was extracted from the mouse faeces using an E.Z.N.A. Stool DNA kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. PCR amplification of the V1-V3 region of bacterial 16s rDNA was performed using universal primers (27F 5′-AGAGTTTGATCCTGGCTCAG-3′, 533R 5′-TTACCGCGGCTGCTGGCAC-3′) incorporating the FLX Titanium adaptors and a barcode sequence. The PCR amplification programme consisted of an initial denaturation step, followed by 25 cycles (annealing temperature at 55 °C), and a final extension step. Equal concentrations of amplicons were pooled from each sample. Emulsion PCR and sequencing were performed according to the recommendations of 454 Life Sciences, on a Roche Genome Sequencer GS FLX Titanium platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.

Fecal pellets were collected from groups of 10–12 Il10rb^-/- and littermate Il10rb^+/- mice starting at day 17 and then every other day until day 28, followed by every week until 14 weeks. Samples were snap frozen and DNA was subsequently extracted by using PowerSoil DNA Isolation Kit (MoBio/Qiagen, Carlsbad, CA) according to the manufacturer’s instructions. Pyrosequencing was performed as described in detail (Wang et al., 2015 (link)) with some modifications. Briefly, the V4 region of the 16S rRNA gene was amplified by PCR using bar-coded universal primers 354F and 926R (Invitrogen/ ThermoFisher Scientific, Waltham, MA). DNA emulsion PCR and 454 DNA sequencing were performed at the BCM Human Genome Sequencing Center in Houston, TX. Sequencing was performed using the 454/Roche B sequencing primer kit in the Roche Genome Sequencer GS-FLX Titanium platform.