Metagenomic Pyrosequencing and Microbial Identification

Overview

Journal Clin Chem

Publisher Oxford University Press

Specialty Biochemistry

Date 2009 Mar 7

PMID 19264858

Citations 166

Authors

Joseph F Petrosino

Sarah Highlander

Ruth Ann Luna

Richard A Gibbs

James Versalovic

Affiliations

Soon will be listed here.

Abstract

Background: The Human Microbiome Project has ushered in a new era for human metagenomics and high-throughput next-generation sequencing strategies.

Content: This review describes evolving strategies in metagenomics, with a special emphasis on the core technology of DNA pyrosequencing. The challenges of microbial identification in the context of microbial populations are discussed. The development of next-generation pyrosequencing strategies and the technical hurdles confronting these methodologies are addressed. Bioinformatics-related topics include taxonomic systems, sequence databases, sequence-alignment tools, and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is summarized with respect to potential pyrosequencing applications.

Summary: Both the approach of 16S rDNA amplicon sequencing and the whole-genome sequencing approach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to tackle such vast amounts of microbiological sequence diversity. Metagenomics, or genetic studies of microbial communities, may ultimately contribute to a more comprehensive understanding of human health, disease susceptibilities, and the pathophysiology of infectious and immune-mediated diseases.

Citing Articles

The Influence of Microbiota on Wild Birds' Parental Coprophagy Behavior: Current Advances and Future Research Directions.

Gul S, Shi Y, Hu J, Song S Microorganisms. 2025; 12(12.

PMID: 39770671 PMC: 11677090. DOI: 10.3390/microorganisms12122468.

The changes of intestinal flora and metabolites in atopic dermatitis mice.

Wang F, Wang Z, Qu L Front Microbiol. 2024; 15:1462491.

PMID: 39736988 PMC: 11683101. DOI: 10.3389/fmicb.2024.1462491.

Characterization of the Blood Bacterial Microbiota in Lowland Tapirs (), a Vulnerable Species in Brazil.

Mongruel A, Medici E, Machado R, Clay K, Andre M Microorganisms. 2024; 12(11).

PMID: 39597659 PMC: 11596849. DOI: 10.3390/microorganisms12112270.

Innumerable Culture-Negative Intracranial Abscesses in an Immunocompetent Young Male Diagnosed by 16s rRNA Gene Sequencing.

Miller R, Kim Y, Pryshliak V, Beutler T Cureus. 2024; 16(10):e71475.

PMID: 39544588 PMC: 11560407. DOI: 10.7759/cureus.71475.

Advancing quantitative PCR with color cycle multiplex amplification.

Chen W, Zhang K, Huang F, Zhao L, Waldren G, Jiang Q Nucleic Acids Res. 2024; 52(17):e81.

PMID: 39119904 PMC: 11417387. DOI: 10.1093/nar/gkae683.

References

DeSantis T, Dubosarskiy I, Murray S, Andersen G . Comprehensive aligned sequence construction for automated design of effective probes (CASCADE-P) using 16S rDNA. Bioinformatics. 2003; 19(12):1461-8. DOI: 10.1093/bioinformatics/btg200. View

Yooseph S, Sutton G, Rusch D, Halpern A, Williamson S, Remington K . The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families. PLoS Biol. 2007; 5(3):e16. PMC: 1821046. DOI: 10.1371/journal.pbio.0050016. View

Pruesse E, Quast C, Knittel K, Fuchs B, Ludwig W, Peplies J . SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007; 35(21):7188-96. PMC: 2175337. DOI: 10.1093/nar/gkm864. View

Thies F, Konig W, Konig B . Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting. J Med Microbiol. 2007; 56(Pt 6):755-761. DOI: 10.1099/jmm.0.46562-0. View

Hunt J, Boddy L, Randerson P, Rogers H . An evaluation of 18S rDNA approaches for the study of fungal diversity in grassland soils. Microb Ecol. 2004; 47(4):385-95. DOI: 10.1007/s00248-003-2018-3. View

Goldberg S, Johnson J, Busam D, Feldblyum T, Ferriera S, Friedman R . A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes. Proc Natl Acad Sci U S A. 2006; 103(30):11240-5. PMC: 1544072. DOI: 10.1073/pnas.0604351103. View

Olsen G, Matsuda H, Hagstrom R, Overbeek R . fastDNAmL: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput Appl Biosci. 1994; 10(1):41-8. DOI: 10.1093/bioinformatics/10.1.41. View

Allander T, Tammi M, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B . Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U S A. 2005; 102(36):12891-6. PMC: 1200281. DOI: 10.1073/pnas.0504666102. View

Wang D, Coscoy L, Zylberberg M, Avila P, Boushey H, Ganem D . Microarray-based detection and genotyping of viral pathogens. Proc Natl Acad Sci U S A. 2002; 99(24):15687-92. PMC: 137777. DOI: 10.1073/pnas.242579699. View

10.

Wheeler D, Srinivasan M, Egholm M, Shen Y, Chen L, McGuire A . The complete genome of an individual by massively parallel DNA sequencing. Nature. 2008; 452(7189):872-6. DOI: 10.1038/nature06884. View

11.

Schloss P, Handelsman J . Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol. 2005; 71(3):1501-6. PMC: 1065144. DOI: 10.1128/AEM.71.3.1501-1506.2005. View

12.

Yu Y, Breitbart M, McNairnie P, Rohwer F . FastGroupII: a web-based bioinformatics platform for analyses of large 16S rDNA libraries. BMC Bioinformatics. 2006; 7:57. PMC: 1386709. DOI: 10.1186/1471-2105-7-57. View

13.

Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar . ARB: a software environment for sequence data. Nucleic Acids Res. 2004; 32(4):1363-71. PMC: 390282. DOI: 10.1093/nar/gkh293. View

14.

Ronaghi M . Improved performance of pyrosequencing using single-stranded DNA-binding protein. Anal Biochem. 2000; 286(2):282-8. DOI: 10.1006/abio.2000.4808. View

15.

Sarkar I, Egan M, Coruzzi G, Lee E, DeSalle R . Automated simultaneous analysis phylogenetics (ASAP): an enabling tool for phlyogenomics. BMC Bioinformatics. 2008; 9:103. PMC: 2270260. DOI: 10.1186/1471-2105-9-103. View

16.

Yang Z, Tu M, Liu J, Wang X, Jin H . Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B. World J Gastroenterol. 2006; 12(44):7192-6. PMC: 4087785. DOI: 10.3748/wjg.v12.i44.7192. View

17.

Zoetendal E, Vaughan E, de Vos W . A microbial world within us. Mol Microbiol. 2006; 59(6):1639-50. DOI: 10.1111/j.1365-2958.2006.05056.x. View

18.

Tarnberg M, Jakobsson T, Jonasson J, Forsum U . Identification of randomly selected colonies of lactobacilli from normal vaginal fluid by pyrosequencing of the 16S rDNA variable V1 and V3 regions. APMIS. 2003; 110(11):802-10. DOI: 10.1034/j.1600-0463.2002.1101106.x. View

19.

Suau A, Bonnet R, Sutren M, Godon J, Gibson G, Collins M . Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol. 1999; 65(11):4799-807. PMC: 91647. DOI: 10.1128/AEM.65.11.4799-4807.1999. View

20.

Prindiville T, Cantrell M, Wilson K . Ribosomal DNA sequence analysis of mucosa-associated bacteria in Crohn's disease. Inflamm Bowel Dis. 2005; 10(6):824-33. DOI: 10.1097/00054725-200411000-00017. View