454 life sciences genome sequencer flx machine

Extracted gDNA was amplified using primers targeting the V1 to V3 regions of the 16S rRNA gene. For bacterial gDNA amplification, barcoded primers of 27F 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-MID-GAGTTTGATCMTGGCTCAG-3′, which was consisted of the GS FLX+ adapter sequence A (underlined sequence), linker nucleotides (bolded sequence), multiplex identifiers sequence (MID), and the universal 16S rRNA-specific sequence (italic sequence). The reverse primer 518R 5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-MID-WTTACCGCGGCTGCTGG-3′ consisted of the GS FLX+ adapter sequence B (underlined sequence), linker nucleotides (bolded sequence), MID, and the universal 16S rRNA-specific sequence (italic sequence). The amplifications were performed at the following conditions: initial denaturation at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 95 °C for 40 seconds, primer annealing at 57 °C for 40 seconds, and extension at 72 °C for 60 seconds; followed by a final elongation at 72 °C for 60 seconds. 16S rRNA PCR products were quantified, pooled, and purified for the sequencing reaction. Sequencing was performed using a 454 Life Sciences Genome Sequencer FLX+ machine (Roche, Florence, SC, USA) according to the manufacturer’s instructions.

Pooled amplicons were purified using the E.Z.N.A^® Cycle Pure Kit (OMEGA, Bio-tek) and Cycle-Pure Spin Protocol according to the manufacturer’s instructions. Sequencing was conducted at the Lund University Sequencing Facility, Sweden. Pooled amplicons were reduced for short fragments by using Agencourt AMPure XP (Beckman Coulter) and inspected using a DNA 1000 kit on a 2100 Bioanalyzer (Agilent). Amplicons were quantified using the Quant-iT dsDNA assay kit (Invitrogen) and Quantifluor fluorometer (Promega), and pools were diluted to obtain a total of 1×10⁷ copies μL⁻¹. Titration and library production (aiming at 10–15% enrichment) were performed using emulsion PCR and the Lib-A kit (Roche). DNA-positive beads were enriched, counted on an Innovatis CASY particle counter (Roche), processed using the XLR70 sequencing kit (Roche), and loaded onto a picotiter plate for pyrosequencing on a 454 Life Sciences Genome Sequencer FLX machine (Roche). DNA sequences were archived at NCBI SRA under the accession number SRP039011.