16S rRNA of female endometrial samples of patients with endometrial cancer and benign gynecologic conditions and methodological controls
The quality of the extracted DNA samples was checked using Qubit Fluorometers (ThermoFisher). The 16S rRNA amplicon libraries were constructed using the Earth Microbiome Project (EMP) primers (515F-806R) with Golay barcode tags on the forward primer.76 (PCR mix ratio is shown in Table 1.) The annealing was performed for 20 seconds at 50C. The extension of the 16S rRNA fragment was performed for 45 seconds at 72C. The PCR for both vaginal and rectal samples used 30 cycles. The quality of amplicons was checked via agarose gel electrophoresis. The amplicon library of the samples that did not pass the quality check was reconstructed until they passed the quality check. The concentration of the quality-assured amplicons was measured using a fluorescent-based DNA measuring technique, Quant-iT dsDNA High-Sensitivity Assay (Invitrogen).  200 nM of each amplicon was pooled into two library pools for sequencing. The quality of the pool was verified with the Bioanalyzer DNA 1000 chip (Agilent Technologies, Santa Clara, CA), then the pool was combined with 1% PhiX for sequencing. The pools were sequenced on the Illumina MiSeq using the 600-cycle MiSeq reagent kit version 3 (Illumina, San Diego, CA) using the Illumina MiSeq benchtop sequencing platform.
SUB15977868
294P