title: Sequencing of the variable region 4 (V4) of the 16S rRNA gene of microbial communities from deep-sea hydrothermal vents
design: For amplifying the hypervariable V4 region of the SSU rRNA gene of Archaea, we used as forward primer oligonucleotides that contained a modified U519F primer sequence fused to 6-nucleotide key tags and to the 454 FLX sequencing primer A (5’GCCTCCCTCGCGCCATCAGxxxxxxCAGYMGCCRCGGKAAHACC, where the x region represents the key tag and the SSU rRNA primer is bold).  To allow multiplex sequencing, ten such primers were synthesized (Integrated DNA Technologies; HPLC purified), each with a different key tag.  The reverse oligonucleotide primer represented a fusion of the 454 FLX sequencing primer B and a modified Arch806R primer (5’-GCCTTGCCAGCCCGCTCAGGGACTACNSGGGTMTCTAAT, where the 16S rRNA region is bold).  PCR reactions (50-µl volume) contained 300nM of each of forward and reverse primers, 20-50 ng template DNA and were performed using Platinum Taq High Fidelity polymerase (Invitrogen, Carlsbad, CA) using the thermal profile 95oC for 2 min followed by 27 cycles of denaturation at 95oC for 15 sec, primer annealing at 53oC and extension at 68oC for 45 sec, with final extension of 68oC for 3 min.  For a few samples the amount of template DNA was limiting and additional 3-5 PCR cycles were necessary in order to obtain a sufficient product.  Negative control reactions without template were always performed.  The bacterial hypervariable V4 region of the SSU rRNA gene was amplified using a similar approach, using as forward primer key-tagged oligonucleotides 5’GCCTCCCTCGCGCCATCAGxxxxxxAYTGGGYDTAAAGNG3’) and a mix of reverse primers (5’GCCTTGCCAGCCCGCTCAG:TACCRGGGTHTCTAATCC, :TACCAGAGTATCTAATTC, :CTACDSRGGTMTCTAATC and :TACNVGGGTATCTAATCC3’ in a 6:1:2:12 ratio, respectively), designed to cover most of the Bacteria domain.  The amplification protocol was the same as for Archaea except that the annealing temperature was 55oC.  The amplicon products were purified using AMPure paramagnetic beads (Agencourt Bioscience Corporation, Beverly, MA) followed by analysis of their concentration and size using DNA 1000 chips on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Waldbronn, Germany).  Amplicon libraries were prepared for unidirectional sequencing using the emPCR Kit II (Roche) followed by sequencing on a 454 Life Sciences Genome Sequencer FLX (Roche Diagnostics, Indianapolis, IN).  Based on the use of identifying key tags, twenty individual samples were pooled and sequenced on each of the two sides of the picotiter plate.
attributes.tag: ATGCTC