PacBio_validation_for_full_length_bacterial_16S_gene

Background Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S gene. This short read approach can introduce biases depending on which variable regions are used, and has limited resolution. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16s rRNA sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate the latest PacBio P6 sequencing chemistry using three approaches: 1) sequencing the 16S gene from a single bacterial species Staphylococcus aureus to analyse error modes and to optimize the bioinformatics pipeline for further analyses 2) sequencing from a pool of 50 different bacterial colonies from human stool samples to compare with full-length capillary sequence of the 16S genes and 3) sequencing of 16S genes from 11 vaginal microbiome samples and comparing the full-length V1V9 sequence and in silico selected V1V2 gene region with 16S V1V2 regions sequenced using the Illumina MiSeq from the same samples. Results Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.14% on the Staphylococcus aureus 16S gene. Full-length capillary sequencing of the 16S genes from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length 16S gene sequencing. Analysis of the human vaginal microbiome using the V1V2 region on MiSeq generated 129 OTUs from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected V1V2 region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length V1V9 datasets generated 261 OTUs which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index measurement reported an overall higher diversity in the MiSeq dataset. Conclusion The PacBio sequencing error rate is now in the acceptable range of the previously widely-used Roche 454 sequencing platform. Species-level microbiota analysis revealed some inconsistency between full-length bacterial 16S capillary sequencing and PacBio sequencing. A higher bacterial species diversity was detected in the MiSeq datasets.