16S of female vaginal and rectal samples of patients with chronic pelvic pain with or without endometriosis and surgical controls
DNA from clinician-collected vaginal and rectal swabs underwent 16S rRNA gene sequencing. The 16S rRNA gene was amplified using the V4 region, with MyFi Mix (Bioline Meridian, Cat No. BIO-25050). Golay barcode-tagged forward primer and Earth Microbiome Project (EMP) primers (515F-806R) for reverse primers and sequenced on the Illumina MiSeq benchtop sequencing platform (Illumina, San Diego, CA). The PCR was performed on LightCycler 96 (Roche). Amplicons were quantified using Quant-It PicoGreen dsDNA Assay kit (ThermoFisher Scientific, Cat No. P7589), according to the manufacturers protocol. Equal amounts of 240 ng of amplified DNA from each sample were pooled and cleaned using UltraClean PCR Clean-Up Kit (QIAquick PCR Purification Kit, Qiagen, Cat No. 28104). The library was sequenced at the PANDA Core for Genomics and Microbiome Research at the University of Arizona, Steele Childrens Research Centre on MiSeq platform (Illumina).