pyrosequencing of human upper GI tract microbiome: adult male stomach: Sample P2* stomach antrum brush
"The bacterial composition in the upper GI tract was characterized in a subset of participants (n=12) of the Seattle Barrett's Esophagus Research cohort using broad-range 16S PCR and pyrosequencing of biopsy and brush samples collected from squamous esophagus, Barrett's esophagus, stomach corpus and stomach antrum. Three of the individuals were sampled at two separate time points. Each biopsy and brush sample was collected into a cryovial containing media and frozen at -80C until further processing. Total genomic DNA was then isolated using UltraClean(r) Microbial DNA Isolation Kit [MoBio, Carlsbad, CA] according to manufacturer's instructions. Mock digests containing only the media were used to assess contamination from extraction reagents. DNA extracted from biopsy and brush samples was eluted in UltraClean(r) elution buffer. The V3-V4 16S rRNA hypervariable region was amplified in each study sample using broad-range PCR with 347F and 806R primers. Three GS FLX+ Titanium forward primers (Roche, Basel, Switzerland) were used in combination to enrich for a broad range of bacterial taxa. Forward primers F1, F2 and F3 were used in a 3:1:1 ratio (F1: 5'-CGTATCGCCTCCCTCGCGCCATCAGGCggaggcagcagtrrggaat-3'; F2: 5'-CGTATCGCCTCCCTCGCGCCATCAGGCggtggctgcagtrrgg-3'; F3: 5'-CGTATCGCCTCCCTCGCGCCATCAGGCggtggcagcagtrrgg-3'), where the uppercase sequence corresponds to the 454 Life Sciences FLX adapter and lowercase sequence, the target specific 16S rRNA sequence. A Titanium reverse primer (5'-CTATGCGCCTTCCAGCCCGCTCAGXXXXXXGCggactaccvgggtatctaat-3'), containing an adapter sequence, 6bp barcode (represented by X) and a GC linker sequence, was added to each sample to allow for multiplexing the sequencing reactions. Amplified 16S rRNA fragments were PCR purified using Agencourt AMPure beads (Beckman Coulter, Pasadena, CA) and quantified using a Quant-iTTM PicoGreen(r) dsDNA assay kit (Invitrogen). Samples were diluted to 1 x 107 molecules/ml, pooled (16 samples per pool) and sequenced on a 454 FLX or GS Junior instrument (Roche)."
Fred Hutchinson Cancer Research Center
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