title: Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.
abstract: BACKGROUND
  Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq.
  
  
  RESULTS
  Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (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 bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset.
  
  
  CONCLUSIONS
  The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, Cambridge, UK. jw22@sanger.ac.uk.
authors.forename: Josef
authors.initials: J
authors.lastname: Wagner
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, Cambridge, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Coupland
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, Cambridge, UK.
authors.forename: Hilary P
authors.initials: HP
authors.lastname: Browne
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, Cambridge, UK.
authors.forename: Trevor D
authors.initials: TD
authors.lastname: Lawley
authors.affiliation: Tropical Epidemiology Group, London Schools of Hygiene and Tropical Medicine, London, UK.
authors.forename: Suzanna C
authors.initials: SC
authors.lastname: Francis
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, Cambridge, UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
mesh_terms: D001483:Base Sequence; D044822:Biodiversity; D019295:Computational Biology / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D006801:Humans / Q000382:microbiology; D054892:Metagenome; D064307:Microbiota / Q000235:genetics; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000145:classification* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics; D014621:Vagina / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: Capillary sequencing; Full-length bacterial 16S rRNA gene sequencing; MiSeq sequencing; PacBio sequencing