Abstract

Background

The main tool to discover novel microbial eukaryotes is the rRNA approach. This approach has important biases, including PCR discrimination against certain rRNA gene species, which makes molecular inventories skewed relative to the source communities. The degree of this bias has not been quantified, and it remains unclear whether species missed from clone libraries could be recovered by increasing sequencing efforts, or whether they cannot be detected in principle. Here we attempt to discriminate between these possibilities by statistically analysing four protistan inventories obtained using different general eukaryotic PCR primers.

Results

We show that each PCR primer set-specific clone library is not a sample from the community diversity but rather from a fraction of this diversity. Therefore, even sequencing such clone libraries to saturation would only recover that fraction, which, according to the parametric models, varies between 17 ± 4% to 49 ± 10%, depending on the set of primers. The pooled data is thus qualitatively richer than individual libraries, even if normalized to the same sequencing effort.

Conclusion

The use of a single pair of primers leads to significant underestimation of the true community richness at all levels of taxonomic hierarchy. The majority of available protistan rRNA gene surveys likely sampled less than half of the target diversity, and might have completely missed the rest. The use of multiple PCR primers reduces this bias but does not necessarily eliminate it.

Notes

Originally published in BMC Microbiology 8:222 (2008). doi:10.1186/1471-2180-8-222

This study was supported by NSF Grant MCB-0348341 to SSE and by the Deutsche Forschungsgemeinschaft grant STO414/2-1 to TS.

Keywords

rRna, molecular inventories, novel microbial eukaryotes, PCR primers

Subject Categories

Protista - Variation

Disciplines

Microbiology

Publisher

BioMedCentral

Publication Date

12-16-2008

Rights Information

© 2008 Jeon et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Rights Holder

Sunok Jeon, et al.

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