High-resolution phylogenetic microbial community profiling

Singer, Esther; Bushnell, Brian; Coleman‐Derr, Devin; Bowman, Brett; Bowers, Robert M.; Levy, Asaf; Gies, Esther A.; Cheng, Jan‐Fang; Copeland, Alex; Klenk, Hans‐Peter; Hallam, Steven J.; Hugenholtz, Philip; Tringe, Susannah G.; Woyke, Tanja

doi:10.1038/ismej.2015.249

Cited by 240 publications

(220 citation statements)

References 48 publications

(62 reference statements)

Supporting

Mentioning

203

Contrasting

Order By: Relevance

“…Singer and colleagues reported the comparison of PacBio full-length bacterial 16S rRNA gene sequencing with Illumina HiSeq 2500 metagenomic shotgun sequencing and MiSeq bacterial 16S rRNA V4 gene region sequencing using a mock community as well as an environmental sample from Sakinaw Lake, British Columbia [12]. When they compared the PacBio full-length bacterial 16S rRNA gene taxonomic resolution with in silico generated PacBio bacterial 16S rRNA V4 gene region at various taxonomic levels, they were able to classify a higher proportion of phylum, class, family, genus and species level from the full-length bacterial 16S rRNA gene dataset compared to the in silico generated PacBio bacterial 16S rRNA V4 gene region.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 99%

“…However, only a few studies have investigated the use of PacBio technology for the sequencing of the full-length 16S gene [10–12, 14]. Thus, more information is urgently needed to identify the advantages and disadvantages of PacBio CCS for bacterial full-length 16S rRNA gene sequencing.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

View full text Add to dashboard Cite

show abstract

“…'false positive' sequence reads from DNA not known to be present in the mock community DNA, which may result from sample cross contamination in the laboratory or from DNA sequencing errors). The use of mock community DNA can further be used to reveal potential biases associated with both primer sequence selection, and additional biases associated with choices of DNA sequencing platform (Singer et al 2016).…”

Section: Use Of Appropriate Pcr Controlsmentioning

confidence: 99%

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Lear¹,

Dickie²,

Banks³

et al. 2018

107

112

View full text Add to dashboard Cite

Advances in the sequencing of DNA extracted from media such as soil and water offer huge opportunities for biodiversity monitoring and assessment, particularly where the collection or identification of whole organisms is impractical. However, there are myriad methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. To help overcome potential biases that may impede the effective comparison of biodiversity data collected by different researchers, we propose a standardised set of procedures for use on different taxa and sample media, largely based on recent trends in their use. Our recommendations describe important steps for sample pre-processing and include the use of (a) Qiagen DNeasy PowerSoil ® and PowerMax ® kits for extraction of DNA from soil, sediment, faeces and leaf litter; (b) DNeasy PowerSoil ® for extraction of DNA from plant tissue; (c) DNeasy Blood and Tissue kits for extraction of DNA from animal tissue; (d) DNeasy Blood and Tissue kits for extraction of DNA from macroorganisms in water and ice; and (e) DNeasy PowerWater ® kits for extraction of DNA from microorganisms in water and ice. Based on key parameters, including the specificity and inclusivity of the primers for the target sequence, we recommend the use of the following primer pairs to amplify DNA for analysis by Illumina MiSeq DNA sequencing: (a) 515f and 806RB to target bacterial 16S rRNA genes (including regions V3 and V4); (b) #3 and #5RC to target eukaryote 18S rRNA genes (including regions V7 and V8); (c) #3 and #5RC are also recommended for the routine analysis of protist community DNA; (d) ITS6F and ITS7R to target the chromistan ITS1 internal transcribed spacer region; (e) S2F and S3R to target the ITS2 internal transcribed spacer in terrestrial plants; (f) fITS7 or gITS7, and ITS4 to target the fungal ITS2 region; (g) NS31 and AML2 to target glomeromycota 18S rRNA genes; and (h) mICOIintF and jgHCO2198 to target cytochrome c oxidase subunit I (COI) genes in animals. More research is currently required to confirm primers suitable for the selective amplification of DNA from specific vertebrate taxa such as fish. Combined, these recommendations represent a framework for efficient, comprehensive and robust DNA-based investigations of biodiversity, applicable to most taxa and ecosystems. The adoption of standardised protocols for biodiversity assessment and monitoring using DNA extracted from environmental samples will enable more informative comparisons among datasets, generating significant benefits for ecological science and biosecurity applications.

show abstract

“…Third-generation sequencing platforms can provide even greater detail to amplicon-based approaches. For instance, sequencing the entire 16S rRNA gene using PacBio® technology resulted in a higher phylogenetic resolution compared to shorter amplicons which are mostly generated from Illumina sequencing (Singer et al 2016). However, new sequencing approaches often need new protocols and new bioinformatic tools.…”

Section: Discussionmentioning

confidence: 99%

“…Amplicon length is crucial, as longer sequences will considerably increase annotation accuracy and phylogenetic resolution (Singer et al 2016). If libraries will be sequenced using Illumina® Miseq (2 × 300 bp) or HiSeq (2 × 250 bp) paired-end sequencing technology, amplicon sizes up to 550 bp are possible.…”

Section: Primer Designmentioning

confidence: 99%

Analysis of soil microbial communities based on amplicon sequencing of marker genes

Schöler¹,

Jacquiod²,

Vestergaard³

et al. 2017

Biol Fertil Soils

213

View full text Add to dashboard Cite

The use of cultivation independent methods has revolutionized soil biology in the last decades. Most popular approaches are based on directly extracted DNA from soil and subsequent analysis of PCR-amplified marker genes by nextgeneration sequencing. While these high-throughput methods offer novel possibilities over cultivation-based approaches, several key points need to be considered to minimize potential biases during library preparation and downstream bioinformatic analysis. This opinion paper highlights crucial steps that should be considered for accurate analysis and data interpretation.

show abstract

High-resolution phylogenetic microbial community profiling

Cited by 240 publications

References 48 publications

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

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

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Analysis of soil microbial communities based on amplicon sequencing of marker genes

Contact Info

Product

Resources

About