Details zur Publikation

Kategorie Textpublikation
Referenztyp Zeitschriften
DOI 10.1371/journal.pone.0032139
Titel (primär) Diversity measures in environmental sequences are highly dependent on alignment quality—Data from ITS and new LSU primers targeting basidiomycetes
Autor Krüger, D.; Kapturska, D.; Fischer, C.; Daniel, R.; Wubet, T. ORCID logo
Quelle PLoS ONE
Erscheinungsjahr 2012
Department BOOEK
Band/Volume 7
Heft 2
Seite von e32139
Sprache englisch
Daten-/Softwarelinks https://doi.org/10.5061/dryad.4h402262
Keywords subunit ribosomal-rna; gradient gel-electrophoresis; arbuscular mycorrhizal fungi; saccharomyces-cerevisiae; phylogenetic analyses; molecular-identification; microbial communities; nucleotide-sequence; concerted evolution; secondary structure; subunit ribosomal-rna; gradient gel-electrophoresis; arbuscular mycorrhizal fungi; saccharomyces-cerevisiae; phylogenetic analyses; molecular-identification; microbial communities; nucleotide-sequence; concerted evolution; secondary structure
Abstract

The ribosomal DNA comprised of the ITS1-5.8S-ITS2 regions is widely used as a fungal marker in molecular ecology and systematics but cannot be aligned with confidence across genetically distant taxa. In order to study the diversity of Agaricomycotina in forest soils, we designed primers targeting the more alignable 28S (LSU) gene, which should be more useful for phylogenetic analyses of the detected taxa. This paper compares the performance of the established ITS1F/4B primer pair, which targets basidiomycetes, to that of two new pairs. Key factors in the comparison were the diversity covered, off-target amplification, rarefaction at different Operational Taxonomic Unit (OTU) cutoff levels, sensitivity of the method used to process the alignment to missing data and insecure positional homology, and the congruence of monophyletic clades with OTU assignments and BLAST-derived OTU names. The ITS primer pair yielded no off-target amplification but also exhibited the least fidelity to the expected phylogenetic groups. The LSU primers give complementary pictures of diversity, but were more sensitive to modifications of the alignment such as the removal of difficult-to align stretches. The LSU primers also yielded greater numbers of singletons but also had a greater tendency to produce OTUs containing sequences from a wider variety of species as judged by BLAST similarity. We introduced some new parameters to describe alignment heterogeneity based on Shannon entropy and the extent and contents of the OTUs in a phylogenetic tree space. Our results suggest that ITS should not be used when calculating phylogenetic trees from genetically distant sequences obtained from environmental DNA extractions and that it is inadvisable to define OTUs on the basis of very heterogeneous alignments.
dauerhafte UFZ-Verlinkung https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=12165
Krüger, D., Kapturska, D., Fischer, C., Daniel, R., Wubet, T. (2012):
Diversity measures in environmental sequences are highly dependent on alignment quality—Data from ITS and new LSU primers targeting basidiomycetes
PLoS One 7 (2), e32139 10.1371/journal.pone.0032139