DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates

The discovery of a large and flourishing population of Phreodrilidae in terrestrial peatlands in northwest Ireland was surprising on two counts: these oligochaete worms are usually aquatic and most of the species occur in the Southern Hemisphere. The phreodrilids were discovered in a project that targeted Enchytraeidae, therefore methods adapted to the investigation of enchytraeids could be applied, including the study of living animals and properly fixed whole mounts. DNA sequencing was also performed. All worms identified here belong to one species, new to science, and placed in the genus Astacopsidrilus, because of the ventral position of the spermathecal pores and the opening of the female funnels inside the spermathecal vestibule. Astacopsidrilus hibernicus sp. nov. is mainly distinguished by thick segmental cushions of epidermal gland cells on the dorsal side of the posterior body half. Male sexual organs and spermathecae are comparatively small and without the often-observed bizarre modifications common in species of this family. DNA sequencing yielded a fragment of the 16S rRNA gene. This is the first description of a phreodrilid species from Europe; the few previous recordings of this family in Ireland and the United Kingdom had been left unidentified.

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Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

10.7287/peerj.preprints.778v2 ◽

2016 ◽

Cited By ~ 2

Author(s):

Patrick D Schloss ◽

Matthew L Jenior ◽

Charles C. Koumpouras ◽

Sarah L Westcott ◽

Sarah K Highlander

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Sequencing ◽

Error Rate ◽

Full Length ◽

Rrna Genes ◽

Rrna Gene ◽

Mock Community ◽

Sequencing Platforms ◽

The 16S Rrna Gene

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality, but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

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Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

10.7287/peerj.preprints.778 ◽

2016 ◽

Author(s):

Patrick D Schloss ◽

Matthew L Jenior ◽

Charles C. Koumpouras ◽

Sarah L Westcott ◽

Sarah K Highlander

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Sequencing ◽

Error Rate ◽

Full Length ◽

Rrna Genes ◽

Rrna Gene ◽

Mock Community ◽

Sequencing Platforms ◽

The 16S Rrna Gene

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality, but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

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Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

10.7287/peerj.preprints.778v1 ◽

2015 ◽

Cited By ~ 5

Author(s):

Patrick D Schloss ◽

Sarah L Westcott ◽

Matthew L Jenior ◽

Sarah K Highlander

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Sequencing ◽

Error Rate ◽

Sequencing Error ◽

Rrna Gene ◽

Sequencing Data ◽

Mock Community ◽

Sequencing Platforms ◽

The 16S Rrna Gene

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality, but short sequences. These platforms have allowed researchers to significantly improve the design of their experiments. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The synthetic mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 2.16% to 0.32%. Unfortunately, this error rate was still 16-times higher than the error rate that has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the longer reads frequently provided better classification, the wider adoption of this approach for 16S rRNA gene sequencing is likely limited by its high sequencing error and low yield of sequencing data relative to the other available platforms.

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Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

PeerJ ◽

10.7717/peerj.1869 ◽

2016 ◽

Vol 4 ◽

pp. e1869 ◽

Cited By ~ 125

Author(s):

Patrick D. Schloss ◽

Matthew L. Jenior ◽

Charles C. Koumpouras ◽

Sarah L. Westcott ◽

Sarah K. Highlander

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Sequencing ◽

Error Rate ◽

Full Length ◽

Rrna Genes ◽

Rrna Gene ◽

Mock Community ◽

Sequencing Platforms ◽

The 16S Rrna Gene

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

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Accurate Determination of Bacterial Abundances in Human Metagenomes Using Full-length 16S Sequencing Reads

10.1101/228619 ◽

2017 ◽

Cited By ~ 2

Author(s):

Fanny Perraudeau ◽

Sandrine Dudoit ◽

James H. Bullard

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Sequencing ◽

Accurate Determination ◽

Full Length ◽

Sequencing Error ◽

Marker Genes ◽

Rrna Gene ◽

Chain Model ◽

The 16S Rrna Gene

AbstractDNA sequencing of PCR-amplified marker genes, especially but not limited to the 16S rRNA gene, is perhaps the most common approach for profiling microbial communities. Due to technological constraints of commonly available DNA sequencing, these approaches usually take the form of short reads sequenced from a narrow, targeted variable region, with a corresponding loss of taxonomic resolution relative to the full length marker gene. We use Pacific Biosciences single-molecule, real-time circular consensus sequencing to sequence amplicons spanning the entire length of the 16S rRNA gene. However, this sequencing technology suffers from high sequencing error rate that needs to be addressed in order to take full advantage of the longer sequence. Here, we present a method to model the sequencing error process using a generalized pair hidden Markov chain model and estimate bacterial abundances in microbial samples. We demonstrate, with simulated and real data, that our model and its associated estimation procedure are able to give accurate estimates at the species (or subspecies) level, and is more flexible than existing methods like SImple Non-Bayesian TAXonomy (SINTAX).

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P1409 PCR of the 16S rRNA gene and DNA sequencing in establishing the aetiology of bacterial infections in children

International Journal of Antimicrobial Agents ◽

10.1016/s0924-8579(07)71248-7 ◽

2007 ◽

Vol 29 ◽

pp. S392-S393

Author(s):

A. Gleesen ◽

C. Grarup

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Sequencing ◽

Bacterial Infections ◽

Rrna Gene ◽

The 16S Rrna Gene

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Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.052993-0 ◽

2014 ◽

Vol 64 (Pt_3) ◽

pp. 781-786 ◽

Cited By ~ 33

Author(s):

Maximo Sánchez ◽

Martha-Helena Ramírez-Bahena ◽

Alvaro Peix ◽

María J. Lorite ◽

Juan Sanjuán ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Similarity ◽

Carbon Sources ◽

Lotus Corniculatus ◽

Culture Conditions ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

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