#201 : Comparison of the Sensitivity of Detecting Cervical Bacteria with Next Generation Sequencing and Third Generation Sequencing Technologies

Abstract

Background and Aims: In in vitro fertilization (IVF) cycles, some patients suffering with vaginosis showed poor reproductive outcome even transferring with good quality embryos. The aim of this pilot study was to evaluate whether the species of Lactobacillus could be detected by next generation sequencing (NGS) and Third generation sequencing (TGS). Method: 18 Patients aged 32–48 years-old who visited our fertility center from February 2021 to June 2022 with previous failure of transfer cycle were included. Exclusion criteria was antibiotic treatment within 3 months prior to enrolment. Genomic DNA of cervical microbiota taken from Cervico vaginal swabs in all patients were extracted and amplified. NGS was performed following the protocol of Ion 16S Metagenomics Kit by detecting V2–4–8 and V3–6, 7–9 regions of 16S. Amplicons were sequenced with Ion GeneStudio S5 Prime System. For TGS, full length of 16S was sequenced with Single Molecule, Real-Time (SMRT) Sequencing (Pacific Biosciences) and analyzed. Results: All of the 18 cervical samples could be amplified with V2–4–8 and V3–6, 7–9 primers and the genus could be 100% identified by NGS. However, most of the Lactobacillus includes L. crispatus, L. jensenii. and L. gasseri showed indistinguishable except of L. iners. On the other hand, TGS clearly identified all Lactobacillus. For Gardnerella, Atopobium and Prevotella, TGS and NGS showed equivalent sensitivity in genus level. However, due to the sequence similarity, Escherichia/Shigella could not be identified from the Lactobacillus. Conclusion: In this report, we aim to compare the sensitivity of detecting bacteria by 16S amplification method. Preliminary results suggest that NGS can distinguish bacterium causes vaginosis in genus level, but there might be misleading if the existence of pathogen such as Escherichia/Shigella. Till now, TGS still exhibits the best sensitivity for distinguish Lactobacillus in species level.