HEUG2XR01__AAGGCGTA__CYAALGF
DNA extraction from individual sample by a cetyl trimethyl ammonium bromide (CTAB) method.
For each sample, 16S rRNA gene were amplified using the primers CYA_ALG_F fused with the 454 pyrosequencing Adaptor A (5'-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-3') and the 8-mer molecular ID (Hamady et al., 2008) and CYA_ALG_R with a buffer system of Ampdirect Plus (Shimadzu Corp., Kyoto, Japan) and BIOTAQ HS DNA Polymerase (Bioline, London, U.K.). Polymerase chain reaction (PCR) was conducted using a temperature profile of 95 deg C for 10 min, followed by 30 cycles at 94 deg C for 20 sec, 48 deg C for 30 sec, 72 deg C for 30 sec, and a final extension at 72 deg C for 7 min. DNA concentrations were measured using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and were diluted into 4ng/uL by Milli-Q water in each PCR sample. The amplicons were pooled and purified using ExoSAP-IT (GE Healthcare, Little Chalfont, Buckinghamshire, U.K.) and a QIAquick PCR Purification Kit (Qiagen).