Amplification of the V5 – V8 region of the 16S rRNA gene effectively speciates medically important genital tractLactobacillusspecies in the upper female genital tract

Abstract

Amplification of the V5 -V8 region of the 16S rRNA gene effectively speciates medically important 2 genital tract Lactobacillus species in the upper female genital tract 3 4 AUTHORS 5 ABSTRACT 31Background: The endometrial cavity is an upper genital tract site largely heralded as sterile, however, 32 advances in culture-independent, next generation sequencing technology have revealed that this site 33 harbours a rich microbial community which includes multiple Lactobacillus species. These bacteria are 34 considered to be the most common non-pathogenic genital tract commensals. Next-generation 35 sequencing of the female lower genital tract has revealed significant variation amongst microbial 36 community composition with respect to Lactobacillus sp. in samples collected from healthy and 37 diseased women. The aim of this study was to evaluate the ability of the 16S rRNA gene to 38 characterize genital tract lactobacilli to species-level taxonomy. 39Methods: Samples were interrogated for the presence of microbial DNA using two-step next 40 generation sequencing technology to exploit the V5-V8 regions of the 16S rRNA gene and compared 41 to standard speciation using qPCR. 42Results: The V5-V8 region of the 16S rRNA gene has sufficient sequence variation within frequently 43 encountered genital tract lactobacilli to allow accurate determination of relative abundance within 44 the community, and speciation for several key community members without completing additional 45 experimentation. 46 Conclusions: Next-generation sequencing of clinical genital tract isolates is an effective method for 47 high throughput identification to species-level of key Lactobacillus sp. 48 KEYWORDS 49 Lactobacillus sp.; genital tract; 16S rRNA; pyrosequencing; qPCR; speciation 50 51 IMPORTANCE 52

The power of DNA based methods in probiotic authentication

“…Thus, a variable region of 16S rRNA is typically selected for high throughput sequencing. The selection of variable regions of 16S rRNA gene to characterize bacterial communities is important because of the preferential primer binding to the different taxa ( O’Callaghan et al, 2019 ). Thus, sequencing different variable regions can result in different profiles of bacterial communities ( Alcon-Giner et al, 2017 ).…”

“…The V3–V4 region is commonly used for bacterial community characterization from various sources ( Almeida et al, 2019 ; Amrane et al, 2019 ; Chen et al, 2020 ; Shehata et al, 2020 ; Shehata and Newmaster, 2020a ). The V5–V8 region of 16S rRNA gene was found to have sufficient sequence variation to allow accurate species level identification of the genus Lactobacillus ( O’Callaghan et al, 2019 ). The use of the V5–V8 region of 16S rRNA gene in this study resulted in a higher resolution compared to V3–V4 region, where most species were identified to species level ( Figure 2 ).…”

“…Sequencing libraries were prepared from five multi-strain finished products following the protocols in the 16S Metagenomic Sequencing Library Preparation Guide ( Illumina, 2013 ). Both the V3–V4 ( Illumina, 2013 ) and the V5–V8 regions ( O’Callaghan et al, 2019 ) of the 16S rRNA gene were used. First stage PCR primers consisted of locus-specific sequences and overhang nucleotide sequences ( Illumina, 2013 ; O’Callaghan et al, 2019 ).…”

“…Both the V3–V4 ( Illumina, 2013 ) and the V5–V8 regions ( O’Callaghan et al, 2019 ) of the 16S rRNA gene were used. First stage PCR primers consisted of locus-specific sequences and overhang nucleotide sequences ( Illumina, 2013 ; O’Callaghan et al, 2019 ). Each PCR reaction mix (25 μl total volume) contained 1× KAPA HiFi HotStart Ready Mix (KAPA Biosystems, United States), 0.2 μM of each primer (Integrated DNA Technologies, IDT, United States), and 5 μL of DNA.…”