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Pyrosequencing as a next-generation sequencing technology in exploring complex microbial communities in ecosystems

Shameem M. Jauffur

Department of Civil Engineering & Applied Mechanics,
Microbial Community Engineering Laboratory,
McGill University,
845 Sherbrooke St.,
West, Montreal, QC,

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“The role of the infinitely small in nature is infinitely great” opined Louis Pasteur. Indeed, since microbes have inhabited earth, they have played crucial roles in ecosystems. They have been sine qua non to natural biological cycles and systems including the carbon, nitrogen and sulfur cycles. In addition, they have been at the centre of engineered bioprocesses such as biological wastewater treatment and fermentation. However, understanding and dissecting the complex microbial community structure, composition, spatial distribution and dynamics in ecosystems are highly challenging considering the salient limitations of traditional or conventional culture-dependent techniques. Although with the emergence of molecular methods such as Terminal Restriction Fragment Length Polymorphism (TRFLP), Automated rRNA Intergenic Spacer Analysis (ARISA), Random Amplified Polymorphic DNA (RAPD), Denaturing Gradient Gel Electrophoresis, Single Strand Conformation Polymorphism (SSCP), microautoradiography (MAR) and Fluorescent in situ Hybridization (FISH) among others, enormous strides have been leaped towards the understanding of alpha, beta and gamma microbial diversity, still limitations persist in terms of gaining adequate coverage, and application to field-scale environments and ecosystems.  Many molecular methods provide incomplete analysis as they do not capture the entire complexities of microbial communities. In order to unravel the unparalleled taxonomic diversity of microbial communities in ecological niches, high sample throughput and coverage of phylotypes at high as well as at low abundance are highly desirable.

Analysing Complex Microbial Communities Using 454-Pyrosequencing

Community profiling based on the 16S rRNA gene has been the cornerstone since the past two decades and has revealed a massive amount of information about bacterial and archeal diversity and structure. Sanger sequencing of the 16S rRNA from environmental samples, although yielded considerable information on taxonomic diversity, is rather restricted in capturing complete coverage and, is thus insufficient in describing microbial complexities. Next-generation sequencing strategies involve high throughput sequencing and can effectively provide deep insights in complex microbial communities in ecological niches. Pyrosequencing, developed by Roche 454 Life Science, is one such example and being a high-throughput sequencing technique it can generate a huge amount of DNA reads. Recently, it has been successfully applied in dissecting complex microbial environments such as the human gastrointestinal tract, soil, wastewater and marine sediments. Since, its first publication in 1998, pyrosequencing has revolutionized genomics and metagenomics.  Undoubtedly, this real-time sequencing technique is shedding light into the complexities of microbial populations in many types of environment and holds the key to unraveling the diversity of more complex natural and bio-engineered ecosystems.

The Biochemistry Behind Pyrosequencing

Pyrosequencing involves sequencing by synthesis and is based on the detection of pyrophosphate (PPi) during DNA elongation. PCR products are converted to single stranded DNA (ssDNA), and the template is incubated with enzymes, adenosine 5’-phosphosulfate (APS) and luciferin. The four types of dNTPs are added in succession and DNA polymerase catalyses the incorporation of the dNTPs to their complementary base on the ssDNA template. Any dNTP incorporation is accompanied by the release of pyrophosphate (PPi). ATP sulphurylase converts the PPi into ATP thereby providing energy for the luciferase enzyme to break down luciferin, generating light signals which can be detected as peaks. The apyrase enzyme degrades any unincorporated nucleotide and prepares the ground for the addition of the next type of dNTP.    


The Pros and Cons

Pyrosequencing has sparked renewed interest in probing the 16S rRNA in greater depth and assess metagenome and metatranscriptome altogether while also providing a means to elucidating microbial members of the rare biosphere which occur in relatively low abundances. Besides eliminating the use of cloning vectors and library construction, and their associated biases, pyrosequencing can also read through secondary structures and produce vast amount of sequences of up to 100Mb per run. Previously, the GS20 version could only generate reads of 100bp while the later developed version, FLX system is able to provide reads of up to 200-300bp. The newly launched “Titanium” system can sequence regions up to 400-500bp. It is expected that in the near future this technology will further improve its sequencing capacity in terms of generating accurate read lengths. One of the cons of this technology remains the shorter read lengths that it can generate as compared to the conventional Sanger sequencing method. In addition, the error rate incurred during sequencing is higher (0.1-5% per base) as compared to the Sanger method (0.001% per base). But still pyrosequencing provides an attractive avenue to explore complex microbial communities as it couples ability to provide in-depth coverage, is rapid, and allow the simultaneous sequencing of several samples by using specific sample barcodes to reduce cost.

In addition to the sequencing technology itself, various bioinformatics tools have emerged to process and analyze pyrosequenced raw data in silico to generate meaningful information. Software such as the Newbler Assembler and RDP Pyrosequencing Pipeline provides a systematic way of analyzing data to rapidly gain insights into the complex microbial composition and structure in environmental samples. 


The possibility of discovering new groups of microorganism in complex environmental systems without cultivated strains has been accrued with the emergence of next-generation sequencing technologies such as pyrosequencing. Not only it is now possible to resolve highly complex microbiota composition with greater accuracy but also to link microbial community diversity with niche function. This is crucial in our understanding of microbial ecology and will definitely contribute in formulating models to study ecosystem functions. Concurring with Louis Pasteur saying that “the role of the infinitely small in nature is infinitely great”, the availability of pyrosequencing in today’s ecologist toolbox is, indeed, a major asset in exploring this infinity. 

Related Publications

Daigle, D.; Simen, B. B.; Pochart, P., (2001) 'High-Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems'. In Current Protocols in Molecular Biology, John Wiley & Sons, Inc.: 2001.

Ngom-Bru, C.; Barretto, C., (2012) 'Gut microbiota: methodological aspects to describe taxonomy and functionality'. Briefings in Bioinformatics 2012.

Rea, M. C.; O'Sullivan, O.; Shanahan, F.; O'Toole, P. W.; Stanton, C.; Ross, R. P.; Hill, C., (2012) 'Clostridium difficile Carriage in Elderly Subjects and Associated Changes in the Intestinal Microbiota'. Journal of Clinical Microbiology 2012, 50 (3), 867-875.

Reddy, B. V. B.; Kallifidas, D.; Kim, J. H.; Charlop-Powers, Z.; Feng, Z.; Brady, S. F.,(2012)  'Natural Product Biosynthetic Gene Diversity in Geographically Distinct Soil Microbiomes'. Applied and Environmental Microbiology 2012, 78 (10), 3744-3752.

Zheng, Z.; Advani, A.; Melefors, O.; Glavas, S.; Nordstrom, H.; Ye, W.; Engstrand, L.; Andersson, A. F.,(2011) 'Titration-free 454 sequencing using Y adapters'. Nat. Protocols 2011, 6 (9), 1367-1376.

Microbial Growth in Drinking Water Distribution Systems - Dirk van der Kooij and Paul W.J.J. van der Wielen
 Publication Date: Jun 2013 - ISBN - 9781780400402

Detection of Pathogens in Water Using Micro and Nano-Technology - Giampaolo Zuccheri and Nikolaos Asproulis
 Publication Date: Aug 2012 - ISBN - 9781780401089


Bartram, A. K.; Lynch, M. D. J.; Stearns, J. C.; Moreno-Hagelsieb, G.; Neufeld, J. D.,(2011) 'Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads'. Applied and Environmental Microbiology 2011, 77 (11), 3846-3852.

Claesson, M. J.; Wang, Q.; O'Sullivan, O.; Greene-Diniz, R.; Cole, J. R.; Ross, R. P.; O'Toole, P. W.,(2010) 'Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions'. Nucleic Acids Research 2010, 38 (22), e200.

Royo, J. L.; Hidalgo, M.; Ruiz, A., (2007) 'Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping'. Nat. Protocols 2007, 2 (7), 1734-1739.

Van den Bogert, B.; de Vos, W. M.; Zoetendal, E. G.; Kleerebezem, M., Microarray (2011) 'Analysis and Barcoded Pyrosequencing Provide Consistent Microbial Profiles Depending on the Source of Human Intestinal Samples'. Applied and Environmental Microbiology 2011, 77 (6), 2071-2080.

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