Full-length multiplexed 16S rRNA gene sequencing works well in self-collected decade old cervicovaginal fluid on paper cards

The vaginal microbiota can be profiled based on sequencing the bacterial 16S rRNA gene. Here, we used long-read 16S sequencing in dried cervicovaginal fluid deposited on paper cards that have been stored for up to eleven years at room temperature. Self- and midwife-collected patient samples as well as positive and negative controls were sequenced using the PacBio Revio® system. All samples yielded PCR products of the expected size, around 1500 bp, and no decrease in read quality was observed for cards that have been stored at for eleven years compared to eight years. All expected bacteria in the positive controls were resolved at species level in both wet and dried format indicating no major effects of drying. The microbiota of the self-collected samples has previously been characterized with short-read sequencing of seven hypervariable regions. Comparing the results showed highly similar bacterial distributions and community state types indicating that long-term storage at room temperature only had a small impact on the DNA stability. Mass-spectrometry analysis revealed that a majority of bacterial species detected on DNA-level could also be identified on protein-level. These results holds promise for future applications of full-length 16S rRNA gene sequencing in self-collected and room-temperature stored samples.