Microbial Communities and Metabolites of Rumens from Various Ruminants using a Barcoded Pyrosequencing and 1H NMR Spectroscopy
Total genomic DNA from the rumen pellets for barcoded pyrosequencing was extracted using a Fast-DNA Spin Kit (MPbio) according to the manufacturer’s instructions. Bacterial and archaeal 16S rRNA genes containing hypervariable regions (V1 to V3) were amplified using two primer sets, Bac9F (5'-adaptor B-AC-GAG TTT GAT CMT GGC TCA G-3')/Bac541R (5'-adaptor A-X-AC-WTT ACC GCG GCT GCT GG-3') and Arc21F (5'-adaptor B-GA-TCC GGT TGA TCC YGC CGG-3')/Arc519R (5'-adaptor A-X-GA-GGT DTT ACC GCG GCK GCT G-3') (10, 16, 48, 55), where X denotes unique 7~11 barcode sequences inserted between the 454 Life Sciences adaptor A sequence and the common linkers, AC and GA (Supplementary Table 1). All PCR amplifications were carried out in a 50-µl C1000 thermal cycler (Bio-Rad, USA) containing 1 µl template of genomic DNA, 20 pmol of each primer, and a Taq polymerase mixture (Solgent, Korea) using a cycling regime of 94ºC for 5 min (1 cycle); 94ºC for 30 s, 60ºC (bacteria) or 58ºC (archaea) for 30 s, and 72ºC for 1 min 20 s (30 cycles); and 72ºC for 10 min (1 cycle). The PCR products were purified using a PCR purification kit (Solgent, Korea) for pyrosequencing.