Using pyrosequencing and quantitative PCR to analyze microbial communities

Abstract

New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of the functions than has been possible just a few years ago. In parallel, quantitative real-time polymerase chain reaction (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems are discussed. The parallel sequencing of amplicon libraries and using barcodes to differentiate multiple samples in a pyrosequencing run are explained. The best procedures and chemistries for QPCR amplifications are also described and advantages of applying automation to increase accuracy are addressed. Three examples in which pyrosequencing and QPCR were used together to define and quantify members of microbial communities are provided: in the human large intestine, in a methanogenic digester whose sludge was made more bioavailable by a high-voltage pretreatment, and on the biofilm anode of a microbial electrolytic cell. The key findings in these systems and how both methods were used in concert to achieve those findings are highlighted.

Community fingerprinting in a sequencing world

“…Since the landmark Global Ocean Sampling expedition began in 2003 (Rusch et al ., ), key developments including high‐throughput capabilities, multiplexing technologies and miniaturisation of sequencing chemistries continue to reduce the time and costs involved with sequencing (MacLean et al ., ). The greater affordability and accessibility of next‐generation sequencing (NGS) technologies such as massively parallel pyrosequencing has seen the volume and the application of sequencing dramatically increase from marine, terrestrial and extreme environments, to medical and artificial systems such as wastewater treatment plants (Nowrousian, ; Zhou et al ., ; Novais & Thorstenson, ; Zhang, ). Examination of natural and engineered microbial ecosystems using NGS is now prolific throughout the literature, with the trend likely to continue.…”

Performance and microbial ecology of a nitritation sequencing batch reactor treating high-strength ammonia wastewater

“…The successful application of high-throughput sequencing technologies has made it possible to explore microbial community dynamics with sufficient sequencing depth 14 . Importantly, the high-throughput can make sequencing much more time- and cost-effective 15 16 relative to conventional sequencing methods. Thus, it can facilitate comparative analysis among several samplings.…”

Descrição da microbiota relacionada às transformações do enxofre em sedimentos de manguezais

“…Outra possível razão para a divergência dos dados, possivelmente, são as metodologias empregadas, incluindo o método de extração do DNA e a ferramenta molecular para analisála. Na maioria destes trabalhos empregou-se biblioteca de clones, técnica que, embora permita ter acesso à diversidade dos microrganismos não cultiváveis, gera poucos clones por amostra, o que não permite representar a total extensão da diversidade presente na área (ZHANG, 2011). Estes problemas tentam ser superados com o pirosequenciamento e a metagenômica, os quais são capazes de incrementar a resolução das análises da comunidade microbiana, permitindo conhecer melhor sua complexidade e interações dentro das amostras, além de ajudar a inferir na sua funcionalidade (CARDENAS;TIEDJE, 2008).…”