16S rRNA Amplicon of Human Vagina
Mid-vaginal samples (Copan Eswabs; Copan Diagnostics, Murrieta, CA) were processed for DNA extraction with the MagAttract Microbial DNA Kit (Qiagen, Germantown, MD) using a custom automated protocol on a Microlab Star instrument (Hamilton Company, Reno, NV) following cell lysing via shaking on a TissueLyser (Qiagen, Germantown, MD). The 16S rRNA gene V3-V4 region was amplified and sequenced using a two-step amplification protocol described by Holm et al. The V3-V4 region was targeted in the first amplification step using bacterial primers 338F and 806R. The second amplification step introduced 8-bp dual-index barcodes to the amplicons. Amplicons were pooled, sequenced on a MiSeq or HiSeq instrument (Illumina, San Diego, CA), then demultiplexed as previously described. (Holm JB et al. Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform. mSystems. 2019;4(1).)