454 Sequencing of Intertidal Mixing Zone Fe Barrier-08_C8_RED
"Quantification of bacterial abundance was accomplished through tagged pyrosequencing of the V1-V3 region of the SSU rRNA gene. Extracted genomic DNA (MoBio PowerSoil DNA Isolation Kit) was sent for pyrosequencing at the Research and Testing Laboratory (Lubbock, TX, USA). Samples were amplified for pyrosequencing using a forward and reverse fusion primer. The forward primer was constructed with the Roche A linker (5? CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG 3?), an 8 bp barcode, and the 27F primer (5? GAG TTT GAT CNT GGC TCA G 3?). The reverse fusion primer was constructed with a biotin molecule, the Roche B linker (5? CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG 3?), and the 519R primer (5? GTN TTA CNG CGG CKG CTG 3?). Amplifications were performed in 25 ?l reactions with Qiagen HotStar Taq master mix (Qiagen Inc, Valencia, CA, USA), 1 ?l of each 5 ?M primer, and 1 ?l of template. Reactions were performed on ABI Veriti thermocyclers (Applied Biosytems, Carlsbad, CA, USA) under the following thermal profile: 95?C for 5 min, then 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 visualized with eGels (Life Technologies, Grand Island, NY, USA). Products were then pooled equimolar and each pool was cleaned with Diffinity RapidTip (Diffinity Genomics, West Henrietta, NY, USA), and size selected using Agencourt AMPure XP (BeckmanCoulter, Indianapolis, IN, USA) following Roche 454 protocols (454 Life Sciences, Branford, CT, USA). Size selected pools were then quantified and 150 ng of DNA were hybridized to Dynabeads M-270 (Life Technologies) to create single stranded DNA following Roche 454 protocols (454 Life Sciences). Single stranded DNA was diluted and used in emPCR reactions, which were performed and subsequently enriched. Sequencing followed using established manufacture protocols (454 Life Sciences) on a Roche 454 FLX system with Titanium chemistry (Roche, Nutley, NJ, USA). Processing of raw reads before submission by the Research and Testing pipeline: 1) quality trimming to threshold = 25, 2) removal of singleton reads, 3) chimera checking (UCHIIME), 4) Denoising, and 5) filter to min of 250 bp."
University of Delaware
08_C8_RED