Improved performance of the PacBio SMRT technology for 16S rDNA sequencing

Mosher, Jennifer J.; Bowman, Brett; Bernberg, Erin; Шевченко, О. Г.; Kan, Jinjun; Korlach, Jonas; Kaplan, Louis

doi:10.1016/j.mimet.2014.06.012

Cited by 96 publications

(70 citation statements)

References 2 publications

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“…Improvements in read length, read number (sampling depth), and read quality could help address this challenge by (i) enabling sequencing of several components of or the entire 16S rRNA-ITS-23S rRNA region with low-error amplicon methods (10,74), and (ii) facilitating the dereplication/assembly of shotgun reads generated from community DNA. Improvements in the throughput and quality of single-cell DNA sequencing would provide an ideal solution with its culture independence and genome-wide resolution.…”

Section: Microbial Inheritance As a Tool To Identify Effector Strainsmentioning

confidence: 99%

Identifying strains that contribute to complex diseases through the study of microbial inheritance

Faith¹,

Colombel

Gordon

2015

Proc. Natl. Acad. Sci. U.S.A.

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It has been 35 y since Carl Woese reported in PNAS how sequencing ribosomal RNA genes could be used to distinguish the three domains of life on Earth. During the past decade, 16S rDNA sequencing has enabled the now frequent enumeration of bacterial communities that populate the bodies of humans representing different ages, cultural traditions, and health states. A challenge going forward is to quantify the contributions of community members to wellness, disease risk, and disease pathogenesis. Here, we explore a theoretical framework for studies of the inheritance of bacterial strains and discuss the advantages and disadvantages of various study designs for assessing the contribution of strains to complex diseases.microbial inheritance | strain-resolution human microbial ecology analyses | effector strains | health | disease

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Section: Microbial Inheritance As a Tool To Identify Effector Strainsmentioning

confidence: 99%

Identifying strains that contribute to complex diseases through the study of microbial inheritance

Faith¹,

Colombel

Gordon

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Despite the fact that PacBio platform can provide full-length 16S rRNA sequence data at a fraction of the cost of Sanger sequencing, it is still not as cost-effective as the short-read Illumina or Ion Torrent technologies. This may be one of the reasons why PacBio system has not widely been adopted in microbial community surveys 25, 26 . Here, we demonstrated the advantages of the long-read PacBio sequencing technology by comparing the ability of full-length and partial 16S rRNA sequences in classifying coral-associated bacteria at the species level.…”

Section: Introductionmentioning

confidence: 99%

High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system

Pootakham

Mhuantong

Yoocha

et al. 2017

Sci Rep

106

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Coral reefs are a complex ecosystem consisting of coral animals and a vast array of associated symbionts including the dinoflagellate Symbiodinium, fungi, viruses and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their fundamental roles in fitness and survival of the host animal. The scleractinian coral Porites lutea is one of the dominant reef-builders in the Indo-West Pacific. Currently, very little is known about the composition and structure of bacterial communities across P. lutea reefs. The purpose of this study is twofold: to demonstrate the advantages of using PacBio circular consensus sequencing technology in microbial community studies and to investigate the diversity and structure of P. lutea-associated microbiome in the Indo-Pacific. This is the first metagenomic study of marine environmental samples that utilises the PacBio sequencing system to capture full-length 16S rRNA sequences. We observed geographically distinct coral-associated microbial profiles between samples from the Gulf of Thailand and Andaman Sea. Despite the geographical and environmental impacts on the coral-host interactions, we identified a conserved community of bacteria that were present consistently across diverse reef habitats. Finally, we demonstrated the superior performance of full-length 16S rRNA sequences in resolving taxonomic uncertainty of coral associates at the species level.

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“…The >10-fold increase in the number of OTU clusters obtained from the PacBio platform reflected the large sequencing error rate in the PacBio data at that time. One year later the same group reanalyzed two of their libraries with the updated P4/C2 chemistry and a run time of 180 minutes [11]. The PacBio RS II platform combined with P4/C2 chemistry improved sequence accuracy and number of high quality reads, compared to their initial assessment, from 80% to > 99% for the full length bacterial 16S rRNA gene sequence.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification

et al. 2016

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BackgroundCurrently, 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.ResultsOur 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0891-4) contains supplementary material, which is available to authorized users.

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Improved performance of the PacBio SMRT technology for 16S rDNA sequencing

Cited by 96 publications

References 2 publications

Identifying strains that contribute to complex diseases through the study of microbial inheritance

Identifying strains that contribute to complex diseases through the study of microbial inheritance

High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification

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