SRX262084 LH and DG LiaoheDagang SRP020908 454 pyrosequencing of 16S rRNA gene was performed. A region approximate 526 bp covering V1–V3 region of 16S rRNA gene in each sample were amplified with the primers 27F and 533R containing the A and B adaptors (454 Life Science). The forward primer (B-27F) was 5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGAGAGTTTGATCCTGGCTCAG-3’, and the reverse primer (A-533R) was 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNNNNNTTACCGCGGCTGCTGGCAC-3’, where the B and A adaptor are the sequences in italics and underlined and the Ns represent an ten-base sample unique barcode sequence. The PCRs were performed in triplicate 50 μl reacting system containing 0.6 μM each of the primer, approximate 5 ng of template NDA, 1×PCR buffer, 2.5 U of Pfu DNA Polymerase (MBI. Fermentas, USA). Negative controls were performed without adding template DNA. The amplification program was as follows: an initial denaturation at 94 oC for 4 min, 25 cycles of 94 oC for 30 s, 55 oC for 30 s, 72 oC for 30 s, and a final step of extension of 72 oC for 10 min. After amplification, replicate PCR products of the same sample were pooled and purified using DNA gel extraction kit (Axygen, China). In preparation for sequencing, the concentration of each PCR product was measured using a Quant-iT PicoGreen double-stranded DNA assay (Invitrogen, Germany), and the quality of each PCR product was controlled on an Algilent 2100 bioanalyzer (Agilent, USA). Working pool was a mixture of equimolar rations of each amplicons and subjected to emulsion PCR to generate amplicon libraries, as recommended by 454 Life Science. Amplicon pyrosequencing were performed from the A-end using 454/Roche A sequencing primer kit on a Roche Genome Sequencer GS FLX Titanium platform (Majorbio Bio-Pharm Technology Co., Ltd, Shanghai, China). SRS408949 70_050c_2 RNA-Seq METAGENOMIC unspecified 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX