Abstract
Microbes are ubiquitous and often occur in functionally and taxonomically complex communities. Unveiling these community dynamics is one of the main challenges of microbial research. Combining a robust, cost effective and widely used method such as Terminal Restriction Fragment Length Polymorphism (T-RFLP) with a Next Generation Sequencing (NGS) method (Illumina MiSeq), offers a solid alternative for comprehensive assessment of microbial communities. Here, these two methods were combined in a study of complex bacterial and fungal communities in the nest mounds of the ant Formica exsecta, with the aim to assess the degree to which these methods can be used to complement each other. The results show that these methodologies capture similar spatiotemporal variations, as well as corresponding functional and taxonomical detail, of the microbial communities in a challenging medium consisting of soil, decomposing plant litter and an insect inhabitant. Both methods are suitable for the analysis of complex environmental microbial communities, but when combined, they complement each other well and can provide even more robust results. T-RFLP can be trusted to show similar general community patterns as Illumina MiSeq and remains a good option if resources for NGS methods are lacking.
Highlights
Microbes are the most numerous organisms on earth (Bertrand et al, 2011)
The Good’s estimate of coverage for the terminal restriction fragments (T-RFs) data suggested that 97.9% of the bacterial, and 93% of the fungal diversity had been captured by the method, whereas the coverage for the OTUs was 99.9% and 99.4%, respectively (Table 1)
This study shows that both Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Illumina sequencing are suitable for analysis of the fungal and bacterial communities in nest mounds of ants, but when the two techniques are combined, it can provide an even more robust dataset
Summary
Microbes are the most numerous organisms on earth (Bertrand et al, 2011) Their small size and the difficulty of cultivating many (if not most) species has spurred the development of molecular techniques to study the taxonomy, community structure and functions of natural microbial communities. Several techniques such as Automated Ribosomal Intergenic Spacer Analysis (ARISA) (Ramette, 2009), Temperature Gradient Gel Electrophoresis (TGGE), Denaturing Gradient Gel Electrophoresis (DGGE) (Nocker, Burr & Camper, 2007) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) (Liu et al, 1997; Kent et al, 2003; Cao et al, 2013) have been developed for the assessment of complex microbial communities. To obtain taxonomic identity of microbial community members, T-RFLP is usually combined with time-consuming and expensive preparation and sequencing of clone libraries (Van Elsas & Boersma, 2011)
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