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 Oliver, Alejandro Belanche Gracia, Susan Elizabeth Girdwood, Eric Pinloche, Toby Wilkinson, C. Jamie Newbold*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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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
Pages (from-to)e101435
Number of pages13
JournalPLoS One
Volume9
Issue number7
DOIs
Publication statusPublished - 22 Jul 2014

Keywords

  • DIFFERENT HYPERVARIABLE REGIONS
  • GRADIENT GEL-ELECTROPHORESIS
  • 2 ISONITROGENOUS DIETS
  • RIBOSOMAL-RNA GENES
  • MICROBIAL COMMUNITIES
  • CILIATE PROTOZOA
  • RRS GENES
  • DIVERSITY
  • SHEEP
  • 16S
  • Rumen/microbiology
  • Sheep, Domestic/microbiology
  • Fermentation
  • Sequence Analysis, DNA
  • Microbiota/genetics
  • Ciliophora/physiology
  • Animals
  • Polymorphism, Restriction Fragment Length
  • Base Sequence
  • High-Throughput Nucleotide Sequencing
  • DNA, Bacterial/genetics

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