High-Throughput Next Generation Sequencing

Volume 733 of the series Methods in Molecular Biology pp 143-158


Pyrosequencing of Chaperonin-60 (cpn60) Amplicons as a Means of Determining Microbial Community Composition

  • John Schellenberg
  • , Matthew G. Links
  • , Janet E. Hill
  • , Sean M. Hemmingsen
  • , Geoffrey A. Peters
  • , Tim J. DumonceauxAffiliated withAgriculture and Agri-Food Canada Saskatoon Research Centre Email author 

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The chaperonin-60 universal target (cpn60 UT) is generated from a set of PCR primers and provides a universally conserved, phylogenetically informative sequence signature for determining the composition of microbial communities by DNA sequencing. Pyrosequencing of cpn60 UT amplicons is emerging as a next-generation tool for providing unprecedented sequencing depth and resolution of microbial communities in individual samples. Owing to the increase in sequencing depth, the dynamic range across which the presence and abundance of individual species can be sampled experimentally also increases, significantly improving our ability to investigate microbial community richness and diversity. The flexible format of the pyrosequencing reaction setup combined with the ability to pool samples through the use of multiplexing IDs makes the generation of microbial profiles based on the cpn60 UT both feasible and cost-effective. We describe here the methods we have developed for determining microbial community profiles by pyrosequencing of cpn60 UT amplicons, from generating amplicons to sequencing and data analysis.

Key words

Pyrosequencing Chaperonin-60 cpn60 Microbial communities Metagenomic profiling