Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in microbiome samples

Abstract

Background. Sequencing variable regions of the 16S rRNA gene (≃300bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. Results. DNA from saliva, oral biofilms and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively. With both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in biofilm samples) none of the differences were statistically significant when correcting for multiple testing. Conclusions. The results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Impact of Periodontitis on the Leakage of Oral Bacteria to the Gut

“…DNA isolation was performed using the MagNA Pure LC DNA Isolation Kit III for Bacteria and Fungi (Roche Diagnostics) with additional enzymatic lysis by MagNA Pure LC 2.0 Instrument (Roche Diagnostics) (Buetas et al 2023). After DNA quantification with QubitTM 1X dsDNA HS Assay Kit, the library was prepared and sequenced using the Sequel II Sequencing Kit 2.0 (PacBio) on the Sequel II PacBio system (Callahan et al 2019).…”

“…FL-16S rRNA gene PacBio sequencing was used to obtain higher resolution at species-level annotation (Buetas et al 2023). After quality filtering, a mean of 20,440 reads per sample were obtained, 69.4% of which were assigned to the species level (Appendix Table 3).…”

“…To address this, we conducted a study analyzing the microbial composition of paired saliva, subgingival sulcus, and fecal samples of periodontal patients and oral healthy controls. To allow the identification of oral bacteria in the gut, we used full-length 16S (FL-16S) rRNA gene sequencing, which allows for species- and subspecies-level resolution (Callahan et al 2019; Buetas et al 2023). Through this approach, we aim to identify the co-occurrence of the same species in all 3 niches, thereby enabling us to estimate the degree of ectopic colonization of oral bacteria in the gut.…”

Conservation and diversity of the pollen microbiome of Pan-American maize using PacBio and MiSeq

“…Most prior microbiome reports were limited by short-read 16S rRNA gene amplicons [e.g., hypervariable V4 region (V4-MiSeq)] ( Greay et al, 2019 ; Palkova et al, 2021 ) that lacked the fine resolution captured by the long-read sequencing platform [full-length 16S rRNA gene -PacBio (FL-PacBio)] needed to accurately reveal taxonomic diversity at the species and subspecies level ( Callahan et al, 2019 ). Only a few studies have compared microbiome results from full-length 16S rRNA gene PacBio to partial 16S rRNA gene MiSeq sequencing ( Buetas et al, 2023 ). Furthermore, to the best of our knowledge, the pollen microbiome has only been reported in the literature by using partial 16S rRNA gene sequencing ( Manirajan et al, 2016 , 2018 , 2019 ; Obersteiner et al, 2016 ; Oteros et al, 2018 ; McFrederick and Rehan, 2019 ; Wu et al, 2022 ).…”