Vaginal Microbiome Profiling using Nanopore Sequencing

DNA was isolated from vaginal samples using QIAamp DNA Mini Kit (Qiagen), following the manufacturer’s protocol for Gram-positive and difficult-to-lyse bacteria, and quantified using Qubit 3.0 Fluorometer. 16S rRNA genes in each sample were amplified with the primers, B27F (TTTCTGTTGGTGCTGATATTGCAGRGTTYGATYMTGGCTCAG) and B1492R (ACTTGCCTGTCGCTCTATCTTCRGYTACCTTGTTACGACTT), which were modified from S-D-bact-0008-c-S20 and S-*-Univ-1492-a-A-19.16 (link) DNA libraries, each containing 12 different clinical samples, were prepared using PCR Barcoding Kit - EXP-PBC096 and Ligation Sequencing Kit 1D - SQK-LSK108, and sequenced on MinION MK I with SpotON Flow Cell Mk I FLO-MIN106 R9.4 (Oxford Nanopore Technologies). Sequencing reads were live base called locally using the MinKNOW protocol - NC_48Hr_Sequencing_Run_FLO-MIN106_ SQK-LSK108.py. Sequence data have been submitted to the ENA database with accession number PRJEB41336.