Ion 520 sequencing kits

Sample sequencing was carried out using a fusion-PCR method. Briefly, fusion-primers were designed in accordance with the manufacturer’s guidelines (Ion Amplification Library Preparation – Fusion Method, Life Technologies, Carlsbad, CA) using Ion Xpress Barcodes linked to 16S gene primer pairs targeting hyper-variable regions 1–8 [16 (link)]. Each 25 μl PCR was carried out using: 12.5 μl iQ supermix™ (Bio-Rad, Hercules, CA), 1 μl of both forward and reverse (5 μM) primers, 9.5 μl nuclease-free water and 1 μl of DNA template. DNA from each patient from each sample (uvula swab, proximal esophageal mucosa, distal esophageal mucosa) was used as a template for the creation of subsequent fusion 16S libraries. PCR was completed in a c1000 thermocycler (Bio-Rad) using the following parameters: Cycle 1), 95 C, 3 min, Cycle 2), Step 1—95 C, 45 s; Step 2—Primer-specific annealing temps., 45 s; Step 3—72 C 2:00, repeat 39x; Step 4—72 C for 7:00. PCR products were purified using Qiagen Qiaquick spin-columns and quantified using a spectrophotometer (Bio-Rad). PCR products were then diluted, mixed in equal proportion and sequenced on an Ion Torrent GeneStudio S5 System using Ion 520 sequencing kits together with 520 size chips following the manufacturer’s instructions (Life Technologies).

Sample sequencing was carried out using a fusion-PCR method. Briefly, fusion-primers were designed in accordance with the manufacturer’s guidelines (Ion Amplification Library Preparation – Fusion Method, Life Technologies, Carlsbad, CA) using Ion Xpress Barcodes linked to 16 s gene primer pairs targeting hyper-variable regions 1–8^{13 (link)}. Each 25 µl PCR was carried out using: 12.5 µl iQ supermix^™ (Bio-Rad, Hercules, CA), 1 µl of both forward and reverse (5 µM) primers, 9.5 µl nuclease-free water and 1 µl of DNA template. DNA from each patient from each sample (uvula swab, endoscope swab, proximal esophagus, mid-esophagus, distal esophagus, Barrett’s esophagus) were used as a template for creation of subsequent fusion 16 s libraries. PCR was completed in a c1000 thermocycler (Bio-Rad) using the following parameters: Cycle 1), 95 C, 3 minutes, Cycle 2), Step 1–95 C, 45 seconds; Step 2—Primer-specific annealing temps., 45 seconds; Step 3–72 C 2:00, repeat 39x; Step 4–72 C for 7:00. PCR products were purified using Qiagen Qiaquick spin-columns and quantified using a spectrophotometer (Bio-Rad). PCR products were then diluted, mixed in equal proportion and sequenced on an Ion Torrent GeneStudio S5 System using Ion 520 sequencing kits together with 520 size chips following the manufacturer’s instructions (Life Technologies).