pyrosequencing-based analysis of 16S rRNA genes of sediments of South China Sea
Bacterial 16s rRNA target at the V3 hypervariable region were amplified using a set of primers was designed by adding a 10-nucleotide barcode to the forward primer 8F (5?-AGAGTTTGATCCTGGCTCAG-3?) and reverse primer 533R (5?-TTACCGCGGCTGCTGGCAC-3?). 
Bacterial 16s rRNA target at the V3 hypervariable region were amplified using a set of primers was designed by adding a 10-nucleotide barcode to the forward primer 8F (5?-AGAGTTTGATCCTGGCTCAG-3?) and reverse primer 533R (5?-TTACCGCGGCTGCTGGCAC-3?). The amplification reaction mixture contained 5 Unit of Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), and 1?Pfu reaction buffer, 200 ?M of dNTPs (TaKaRa, Dalin, China), 0.2?M barcoded primer and 20ng genomic DNA template in volume of 100 ?l. PCR was performed with a thermal cycler (Bio-Rad, USA) under the following condition: 5 min at 94?C, 25 cycles of 30 s at 94?C plus 45 s at 55?C plus 30 s at 72?C, and finally 5 min at 72?C.