Evaluation of automated ribosomal intergenic spacer analysis for bacterial fingerprinting of rumen microbiome compared to pyrosequencing technology.

Elie Jami,Itzhak Mizrahi,Naama Shterzer

doi:10.3390/pathogens3010109

Elie Jami, Itzhak Mizrahi + Show 1 more

Open Access

https://doi.org/10.3390/pathogens3010109

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Journal: Pathogens	Publication Date: Jan 22, 2014
Citations: 61	License type: CC BY 3.0

Affiliation: Agricultural Research Organization, Tel Aviv University

Abstract

The mammalian gut houses a complex microbial community which is believed to play a significant role in host physiology. In recent years, several microbial community analysis methods have been implemented to study the whole gut microbial environment, in contrast to classical microbiological methods focusing on bacteria which can be cultivated. One of these is automated ribosomal intergenic spacer analysis (ARISA), an inexpensive and popular way of analyzing bacterial diversity and community fingerprinting in ecological samples. ARISA uses the natural variability in length of the DNA fragment found between the 16S and 23S genes in different bacterial lineages to infer diversity. This method is now being supplanted by affordable next-generation sequencing technologies that can also simultaneously annotate operational taxonomic units for taxonomic identification. We compared ARISA and pyrosequencing of samples from the rumen microbiome of cows, previously sampled at different stages of development and varying in microbial complexity using several ecological parameters. We revealed close agreement between ARISA and pyrosequencing outputs, especially in their ability to discriminate samples from different ecological niches. In contrast, the ARISA method seemed to underestimate sample richness. The good performance of the relatively inexpensive ARISA makes it relevant for straightforward use in bacterial fingerprinting analysis as well as for quick cross-validation of pyrosequencing data.

Highlights

Analysis of bacterial diversity in environmental samples has always been a challenge for microbiologists
automated ribosomal intergenic spacer analysis (ARISA) was used to detect changes in bacterial community composition of a highly complex microbial environment residing in the rumen—the upper digestive tract compartment found in all ruminants [14,15,16,17]
The analysis revealed an overall lower bacterial diversity and a significantly lower average operational taxonomic units (OTUs) number per sample as compared to the pyrosequencing (Table 1), with a total of 341 OTUs detected by ARISA

Summary

Introduction

Analysis of bacterial diversity in environmental samples has always been a challenge for microbiologists. One such popular method, introduced by Fisher and Triplett (1999) [2], is automated ribosomal intergenic spacer analysis (ARISA) This method amplifies the DNA fragment found between the 16S and 23S genes in bacterial genomes and uses its natural variability in length—and separation by a capillary electrophoresis system (such as Sanger sequencing technology)—to infer diversity, with different sizes representing different operational taxonomic units (OTUs). A study by Kovacs et al (2010) [3] revealed that with increasing species diversity, the method tends to underestimate species richness This is the result of the limited fragment lengths that could be detected by this method, which range from 200 to 1,150 bp, restricting the number of different observable phylotypes within a sample to several hundreds. ARISA was used to detect changes in bacterial community composition of a highly complex microbial environment residing in the rumen—the upper digestive tract compartment found in all ruminants [14,15,16,17]

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