Molecular Methods for Studying the Human Microbiota

Yoon Hee Choi; Jin Chung; Hee Sam Na

doi:10.4167/jbv.2013.43.1.67

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Choi, Chung, and Na: Molecular Methods for Studying the Human Microbiota

Letter to the Editor

J Bacteriol Virol 2013;43(1):67-72.

Published online: 9 February 2013

DOI: https://doi.org/10.4167/jbv.2013.43.1.67

Molecular Methods for Studying the Human Microbiota

Yoon Hee Choi¹, Jin Chung², Hee Sam Na^2,^*

¹Department of Hematology-Oncology, Dongnam Institute of Radiological and Medical Sciences, Busan, Korea

²Department of Oral Microbiology, School of Dentistry, Pusan National University, Busan, Korea

^*Corresponding author: Hee Sam Na, Department of Oral Microbiology, School of Dentistry, Pusan National University, Mool Keum, Beomoe Ri, Yangsan, 626-810, Korea. Phone: +82-51-510-8252, Fax: +82-51-510-8246, e-mail: heesamy@pusan.ac.kr

^** This work was supported by a 2-Year Research Grant of Pusan National University.

Received 21 January 201323 January 2013 Accepted 30 January 2013

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Vast array of microbes colonize to each anatomical environment of human body. Culture based methods are important in investigating the microbial structure, but they are extremely biased in their evaluation of microbial diversity by selecting particular population of microbiota. Recent advance in molecular technology has allowed sophisticated analysis of complex human microbiota by culture-independent methods. Here, we will discuss features of tools for human microbiota studies including Roche-454 and Illumina platform. We will also briefly discuss features of some strategies that are commonly applied to these platforms including 16S rRNA targeting and shotgun sequencing. New platforms such as PacBio and Oxford Nanopore are also introduced.

Keywords: Human microbiota, Next-Generation sequencing, Roche-454, Illumina

REFERENCES

1). Kuczynski J, Lauber CL, Walters WA, Parfrey LW, Clemente JC, Gevers D, et al. Experimental and analytical tools for studying the human microbiome. Nat Rev Genet. 2011; 13:47–58.

2). Weinstock GM. Genomic approaches to studying the human microbiota. Nature. 2012; 489:250–6.

3). Consortium HMP. Structure, function and diversity of the healthy human microbiome. Nature. 2012; 486:207–14.

4). Brüls T, Weissenbach J. The human metagenome: our other genome? Hum Mol Genet. 2011; 20:R142–8.

5). Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010; 464:59–65.

6). Bae JW. Recent Methodological Approaches to Human Microbiome. J Bacteriol Virol. 2011; 41:1–7.

7). Olsen GJ, Lane DJ, Giovannoni SJ, Pace NR, Stahl DA. Microbial ecology and evolution: a ribosomal RNA approach. Annu Rev Microbiol. 1986; 40:337–65.

8). Kim W. Application of Metagenomic Techniques: Understanding the Unrevealed Human Microbiota and Explaining the in Clinical Infectious Diseases. J Bacteriol Virol. 2012; 42:263–75.

9). Nelson TA, Holmes S, Alekseyenko AV, Shenoy M, Desantis T, Wu CH, et al. PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity. Neurogastroenterol Motil. 2011; 23:169–77.

10). Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR, et al. Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci U S A. 2006; 103:12115–20.

11). Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, et al. The NIH Human Microbiome Project. Genome Res. 2009; 19:2317–23.

12). Hayashi H, Sakamoto M, Benno Y. Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal bifidobacterium spp. in healthy subjects. Microbiol Immunol. 2004; 48:1–6.

13). Lazarevic V, Whiteson K, Huse S, Hernandez D, Farinelli L, Osterås M, et al. Metagenomic study of the oral microbiota by Illumina high-throughput sequencing. J Microbiol Methods. 2009; 79:266–71.

14). Gloor GB, Hummelen R, Macklaim JM, Dickson RJ, Fernandes AD, MacPhee R, et al. Microbiome profiling by illumina sequencing of combinatorial sequence-tagged PCR products. PLoS One. 2010; 5:e15406.

15). Hummelen R, Fernandes AD, Macklaim JM, Dickson RJ, Changalucha J, Gloor GB, et al. Deep sequencing of the vaginal microbiota of women with HIV. PLoS One. 2010; 5:e12078.

16). Luckey TD. Introduction to intestinal microecology. Am J Clin Nutr. 1972; 25:1292–4.

17). Martin J, Sykes S, Young S, Kota K, Sanka R, Sheth N, et al. Optimizing read mapping to reference genomes to determine composition and species prevalence in microbial communities. PLoS One. 2012; 7:e36427.

18). Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, et al. Enterotypes of the human gut microbiome. Nature. 2011; 473:174–80.

19). Raes J, Foerstner KU, Bork P. Get the most out of your metagenome: computational analysis of environmental sequence data. Curr Opin Microbiol. 2007; 10:490–8.

20). Wooley JC, Godzik A, Friedberg I. A primer on metagenomics. PLoS Comput Biol. 2010; 6:e1000667.

21). Schloss PD, Gevers D, Westcott SL. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One. 2011; 6:e27310.

22). Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R. UniFrac: an effective distance metric for microbial community comparison. ISME J. 2011; 5:169–72.

23). Consortium HMP. A framework for human microbiome research. Nature. 2012; 486:215–21.

24). Abubucker S, Segata N, Goll J, Schubert AM, Izard J, Cantarel BL, et al. Metabolic reconstruction for metagenomic data and its application to the human microbiome. PLoS Comput Biol. 2012; 8:e1002358.

25). Caporaso JG, Lauber CL, Costello EK, Berg-Lyons D, Gonzalez A, Stombaugh J, et al. Moving pictures of the human microbiome. Genome Biol. 2011; 12:R50.

26). Chitsaz H, Yee-Greenbaum JL, Tesler G, Lombardo MJ, Dupont CL, Badger JH, et al. Efficient de novo assembly of single-cell bacterial genomes from short-read data sets. Nat Biotechnol. 2011; 29:915–21.

27). Dichosa AE, Fitzsimons MS, Lo CC, Weston LL, Preteska LG, Snook JP, et al. Artificial polyploidy improves bacterial single cell genome recovery. PLoS One. 2012; 7:e37387.

28). Benson AK, Kelly SA, Legge R, Ma F, Low SJ, Kim J, et al. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proc Natl Acad Sci U S A. 2010; 107:18933–8.

29). Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science. 2012; 336:1268–73.

Table 1.

Comparison of sequencing technologies (modified from ref. 1 and 2)

Platform	Methods	Characteristics	16S rRNA	Shotgun	Relative cost factor (per Mb)	Scale of reads per sample	Raw error rate (%)	Comment
Sanger-based or capillary based instrument	Fluorescent, dideoxy terminator	750-base reads High accuracy	Full length sequenced with 2∼3 reads	Long reads help with database comparisons	100	10²	0.001	Most costly method Relatively low throughput, so low coverage of 16S or shotgun
Roche-454	Pyrosequencing light emission	400-base reads	Up to 3 variable regions per read	Long reads help with database comparisons	1	10³	1	Cost limits shotgun coverage but 16S coverage is good
Illumina	Fluorescent, stepwise sequencing	100∼150 base reads	Only 1 variable region per read	Short reads do not seem to limit analysis	0.1	10⁵∼10⁶	<1	Very high coverage owing to high instrument output and very low cost
PacBio	Fluorescent, single-molecule sequencing	Up to 10-kilobase reads Low accuracy	Accuracy an issue for correct taxon identification	Long reads could help assembly	1.5	10³	15	Attractive for long reads, but lower accuracy limits applications
Ion Torrent	Proton detection	More than 200-base reads	Like other NGS	Like Illumina	0.4	10³	2	Expect high coverage, but longer reads than Illumina

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