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

Fredriksson NJ, Hermansson M, Wilén BM - BMC Bioinformatics (2014)

Bottom Line: Variations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. johan.fredriksson@gu.se.

ABSTRACT

Background: Terminal 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.

Results: Variations 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.

Conclusions: A 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.

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Similarity versus difference in total fluorescence. Bray-Curtis (empty symbols) and Jaccard (filled symbols) similarities of 51 loading duplicates plotted against difference in total fluorescence (relative the highest total fluorescence of the two duplicates) based on peak heights. The T-RF profiles were analyzed with a PDT of 50 (Panel A) and a PDT of 100 (Panel B).
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Fig6: Similarity versus difference in total fluorescence. Bray-Curtis (empty symbols) and Jaccard (filled symbols) similarities of 51 loading duplicates plotted against difference in total fluorescence (relative the highest total fluorescence of the two duplicates) based on peak heights. The T-RF profiles were analyzed with a PDT of 50 (Panel A) and a PDT of 100 (Panel B).

Mentions: In the loading duplicates dataset, the similarities decreased with increasing differences in total fluorescence when a PDT of 50 was applied (Figure 6). The relatively low similarities were due to a high number of T-RFs not observed in both loadings. Generally, both profiles had T-RFs not present in the other, not only the profile with the highest total fluorescence. Most T-RFs that were only observed in one duplicate were of low relative abundance, on average 0.9% ± 1.8%. As a result, the Bray-Curtis similarities, which are calculated using the relative abundances of the T-RFs, were higher than the Jaccard similarities, which put equal weight on all T-RFs (Figure 6). The same pattern was seen when the analysis was based on peak heights as when it was based on peak areas.Figure 6


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

Fredriksson NJ, Hermansson M, Wilén BM - BMC Bioinformatics (2014)

Similarity versus difference in total fluorescence. Bray-Curtis (empty symbols) and Jaccard (filled symbols) similarities of 51 loading duplicates plotted against difference in total fluorescence (relative the highest total fluorescence of the two duplicates) based on peak heights. The T-RF profiles were analyzed with a PDT of 50 (Panel A) and a PDT of 100 (Panel B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4232699&req=5

Fig6: Similarity versus difference in total fluorescence. Bray-Curtis (empty symbols) and Jaccard (filled symbols) similarities of 51 loading duplicates plotted against difference in total fluorescence (relative the highest total fluorescence of the two duplicates) based on peak heights. The T-RF profiles were analyzed with a PDT of 50 (Panel A) and a PDT of 100 (Panel B).
Mentions: In the loading duplicates dataset, the similarities decreased with increasing differences in total fluorescence when a PDT of 50 was applied (Figure 6). The relatively low similarities were due to a high number of T-RFs not observed in both loadings. Generally, both profiles had T-RFs not present in the other, not only the profile with the highest total fluorescence. Most T-RFs that were only observed in one duplicate were of low relative abundance, on average 0.9% ± 1.8%. As a result, the Bray-Curtis similarities, which are calculated using the relative abundances of the T-RFs, were higher than the Jaccard similarities, which put equal weight on all T-RFs (Figure 6). The same pattern was seen when the analysis was based on peak heights as when it was based on peak areas.Figure 6

Bottom Line: Variations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. johan.fredriksson@gu.se.

ABSTRACT

Background: Terminal 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.

Results: Variations 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.

Conclusions: A 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.

Show MeSH