2.5. PCR validation

We experimentally validated bioinformatics findings with de-identified archival tissue from the Hawaii Tumor Registry-Discard Residual Repository (RTR), a unique collection of formalin-fixed, paraffin-embedded (FFPE) tissue from cancer patients diagnosed within the catchment area of the Hawaii Tumor Registry. The Hawaii Tumor Registry is one of three population-based registries associated with the National Cancer Institute (NCI) and the Surveillance, Epidemiology, and End-Results (SEER) program. Archival tissue from a total of 85 paired cases from gastric (21), and colon (64) cancers were selected for validation. Specimen retrieval, cut & slide, sectioning, pathology review, and nucleic acid extraction were performed by the University of Hawaii Cancer Center Pathology Shared Resources. DNA was extracted from FFPE using Qiagen All Prep FFPET Kit (Qiagen, Valencia, CA) and quantified by NanoDrop spectrophotometer (Thermo-Scientific, Wilmington, DE). PCR reactions were completed using 30 ng of DNA for every 25 µl of reaction mix using commercially available species-specific primer-probe kits (Microbial DNA qPCR assay kits 330033, Qiagen, Valencia CA) per manufacturer's instructions under the following conditions: Activation: 10 min 95 °C, followed by 45 cycles of Denaturation and Annealing at 95 °C for 15 s and 60 °C for 2 min. Samples were tested in duplicates plus positive and negative controls. Discrepancies were resolved by repeat qPCR. Species-specific validation was performed for Helicobacter pylori, Bacteroides vulgatus, Fusobacterium nucleatum, and Selenomonas sputigena.