Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments

Jonasson, Jon; Olofsson, Margaretha; Monstein, Hans‐Jürg

doi:10.1034/j.1600-0463.2002.100309.x

Cited by 137 publications

(99 citation statements)

References 17 publications

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“…MPS is also now used to access metagenomes or viruses 7,24,178) or to identify bacteria after PCR (Polymerase Chain Reaction) or MDA (Multiple Displacement Amplification) amplifications 49,50,74,76,85,116,120,129,168) . Parallel sequencing would also have the capability to distinguish a low abundance true pathogen, when increased sensitivity of detection is needed and that low contamination exists in molecular biology reagents, including ultra-pure water 50) .…”

Section: Introductionmentioning

confidence: 99%

Global Sequencing: A Review of Current Molecular Data and New Methods Available to Assess Microbial Diversity

Christen

2008

Microb. Environ.

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Roughly estimating the diversity of life has for a long time been seen as a challenge. Sequences obtained from the environment by direct amplification are now seen as the sole mean to provide information for at least 99% of the prokaryotes in natural communities. Recent massive parallel sequencing technologies now allow to sequence nearly every sequence resulting from such amplification. Are we ready to deal with such a deluge of data? Using three recent publications (16S rRNA genes amplification in soil and seawater followed by massive parallel sequencing) as case examples, this review analyses the state of the field.

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Section: Introductionmentioning

confidence: 99%

Global Sequencing: A Review of Current Molecular Data and New Methods Available to Assess Microbial Diversity

Christen

2008

Microb. Environ.

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“…The nucleotides are added sequentially, and through coupled enzymatic reactions, the polymerase-catalyzed incorporation of nucleotides can be monitored as light peaks in a Pyrogram TM . Pyrosequencing has been used for many applications, such as genotyping of single-nucleotide polymorphisms (17,18 ), mutation detection (19,20 ), and sequence detection (21,22 ). The peak heights in the Pyrogram are directly proportional to the emitted light from the Pyrosequencing reaction.…”

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Determination of CYP2D6 Gene Copy Number by Pyrosequencing

et al. 2005

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Background: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable. Methods: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing TM technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights. Results: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0 -4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high. Conclusions: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

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“…The latter can discriminate a large number of species, but in all of these methods the identification is based on complex band patterns with limitations in resolution of closely related spe- , and noncoding spacer regions as ITS (6). Given the right target, sequencing has the discriminatory power to resolve closely related species, but has limitations in terms of ease of use and cost.An alternative DNA sequencing technique, pyrosequencing, has been shown to accurately identify microorganisms on a large scale in a few hours (22,31). Pyrosequencing is a realtime DNA sequencing method that, on average, analyzes 50 to 60 bases by detecting the release of pyrophosphate.…”

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Identification of 43 Streptococcus Species by Pyrosequencing Analysis of the rnpB Gene

et al. 2005

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Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.Species identification by molecular methods is emerging in the field of microbiological diagnostics and the demand for simple, sensitive, specific, and robust tests is increasing. Broad range identification methods are needed for genera with a large number of species. The genus Streptococcus is a diverse group of approximately 50 species, including many important pathogens for humans and domesticated animals. Current means of classification rely on manual or automated phenotypic test systems, but up to 25% of examined streptococcal strains are incorrectly or ambiguously identified (13, 24). The misidentifications occur because of phenotypic trait variability among strains within a given species (3) and inconsistency in biochemical traits of the same strain (41). Small alterations in manual procedures of the tests may also give false results.Several DNA-based methods have been evaluated for species identification of streptococci. DNA hybridization has been successfully used for detection of few species (4, 18) but has a limited use in broad range identification. Streptococcal species can be identified by measuring the tRNA intergenic lengths (8) and up to 31 species were distinguishable when the fragments were separated by capillary electrophoresis (2). Size separation can also be used for species identification when combined with amplification of selected targets (11, 23), arbitrarily primed PCR (33), and restriction of amplified rRNA genes (21, 37). The latter can discriminate a large number of species, but in all of these methods the identification is based on complex band patterns with limitations in resol...

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Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments

Cited by 137 publications

References 17 publications

Global Sequencing: A Review of Current Molecular Data and New Methods Available to Assess Microbial Diversity

Global Sequencing: A Review of Current Molecular Data and New Methods Available to Assess Microbial Diversity

Determination of CYP2D6 Gene Copy Number by Pyrosequencing

Identification of 43 Streptococcus Species by Pyrosequencing Analysis of the rnpB Gene

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