Archaeal diversity of bulking activated sludge
Archaeal 16S rRNA gene tag-encoded FLX amplicon pyrosequencing (TEFAP): samples were amplified for pyrosequencing using forward and reverse fusion primers. The forward primer was constructed with the Roche A linker, an 8-10 bp barcode and primer 341F (5’-GYGCASCAGKCGMGAAW-3’). The reverse fusion primer was constructed with a biotin molecule, the Roche B linker and primer 958R (5’-GGACTACVSGGGTATCTAAT-3’). Amplifications were performed under the thermal profile: 95°C for 5 min, 35 cycles of 94°C for 30 sec, 54°C for 40 sec, 72°C for 1 min, followed by one cycle of 72°C for 10 min and 4°C hold. Amplification products were pooled equimolar and each pool was cleaned with Diffinity RapidTip and size selected using Agencourt AMPure XP following Roche 454 protocols. Size selected pools were then quantified and 150 ng of DNA were hybridized to Dynabeads M-270 to create single-stranded DNA following Roche 454 protocols. ssDNA was diluted and used in emPCR reactions, which were performed and subsequently enriched. Sequencing was performed following established manufacture protocols (454 Life Sciences).