title: 16S amplicon sequencing of aerobic granular sludge: SBR1 - 30 to 40% sucrose
design: Bacterial 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 28F         (5'-GAGTTTGATCNTGGCTCAG-3'). The reverse fusion primer was constructed with a biotin         molecule, the Roche B linker and primer 519R (5'-GTNTTACNGCGGCKGCTG-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).
identifiers.namespace: SUB1099598
identifiers.id: SBR1_30-40% sucrose