Impact of T-RFLP data analysis choices on assessments of microbial community structure and dynamics.

Nils Johan Fredriksson,Malte Hermansson,Britt-Marie Wilén

doi:10.1186/s12859-014-0360-8

Abstract

BackgroundTerminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. Although the technique is well established there is no consensus on how to treat T-RFLP data to achieve the highest possible accuracy and reproducibility. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples.ResultsVariations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs. A novel method was developed which improved the alignment by adjusting for systematic shifts in the T-RF size estimations between the T-RF profiles. Differences in total fluorescence were shown to be caused by differences in sample concentration and by the gel loading. Five normalization methods were evaluated and the total fluorescence normalization procedure based on peak height data was found to increase the similarity between replicate profiles the most. A high peak detection threshold, alignment correction, normalization and the use of consensus profiles instead of single profiles increased the similarity of replicate T-RF profiles, i.e. lead to an increased reproducibility. The impact of different treatment methods on the outcome of subsequent analyses of T-RFLP data was evaluated using a dataset from a longitudinal study of the bacterial community in an activated sludge wastewater treatment plant. Whether the alignment was corrected or not and if and how the T-RF profiles were normalized had a substantial impact on ordination analyses, assessments of bacterial dynamics and analyses of correlations with environmental parameters.ConclusionsA novel method for the evaluation and correction of the alignment of T-RF profiles was shown to reduce the uncertainty and ambiguity in alignments of T-RF profiles. Large differences in the outcome of assessments of bacterial community structure and dynamics were observed between different alignment and normalization methods. The results of this study can therefore be of value when considering what methods to use in the analysis of T-RFLP data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0360-8) contains supplementary material, which is available to authorized users.

Highlights

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities
DNA fingerprinting, gene sequencing, and in recent years, next-generation sequencing technologies enables descriptions of microbial communities at different resolution, where the latter provides an unprecedented level of detail. Fingerprinting methods, such as terminal restriction fragment polymorphism (T-RFLP) (e.g. [3]), are trumped by nextgeneration sequencing technologies when it comes to describing the depth of microbial communities, numerous studies comparing the two methods have shown that the same conclusions can be drawn from both approaches, both with regard to community structure [4,5,6,7] and dynamics [8,9,10] of the community
We acknowledge that some of the conclusions presented here may be specific for the particular system that we used, since the variations in terminal restriction fragments (T-RFs) abundances and sizes are related to the DNA loading and T-RF detection system

Summary

Introduction

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples. DNA fingerprinting, gene sequencing, and in recent years, next-generation sequencing technologies enables descriptions of microbial communities at different resolution, where the latter provides an unprecedented level of detail. Fingerprinting methods, such as terminal restriction fragment polymorphism (T-RFLP) Despite the continuously decreasing cost and the popularity of next-generation sequencing, fingerprinting techniques such as T-RFLP are still relevant and an important tool for studies of microbial communities

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