Pros and cons of ion-torrent next generation sequencing versus terminal restriction fragment length polymorphism T-RFLP for studying the rumen bacterial community

Gabriel de la Fuente, Alejandro Belanche, Susan E Girwood, Eric Pinloche, Toby Wilkinson, C Jamie Newbold

Research output: Contribution to journalArticle

38 Citations (Scopus)
4 Downloads (Pure)

Abstract

The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T-RFLP). Although absolute number differed, there was a high correlation between NGS and T-RFLP in terms of richness and diversity with R values of 0.836 and 0.781 for richness and Shannon-Wiener index, respectively. Dendrograms for both datasets were also highly correlated (Mantel test = 0.742). Eighteen OTUs and ten genera were significantly impacted by the addition of rumen protozoa, with an increase in the relative abundance of Prevotella, Bacteroides and Ruminobacter, related to an increase in free ammonia levels in the rumen. Our findings suggest that classic fingerprinting methods are still valuable tools to study microbial diversity and structure in complex environments but that NGS techniques now provide cost effect alternatives that provide a far greater level of information on the individual members of the microbial population.

Original languageEnglish
Article numbere101435
JournalPLoS ONE
Volume9
Issue number7
Early online date22 Jul 2014
DOIs
Publication statusFirst published - 22 Jul 2014
Externally publishedYes

Keywords

  • Animals
  • Base Sequence
  • Ciliophora/physiology
  • DNA, Bacterial/genetics
  • Fermentation
  • High-Throughput Nucleotide Sequencing
  • Microbiota/genetics
  • Polymorphism, Restriction Fragment Length
  • Rumen/microbiology
  • Sequence Analysis, DNA
  • Sheep, Domestic/microbiology

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