ArticlePloS one2012
Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 20 papers.
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Who cites it
20 citing papers in PubMed.
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- Mushroom DNA barcoding project: Sequencing a segment of the 28S rRNA gene.Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology · 2020Article
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- Different response of bacteria, archaea and fungi to process parameters in nine full-scale anaerobic digesters.Microbial biotechnology · 2019Article
- Effect of Simulated Climate Warming on the Ectomycorrhizal Fungal Community of Boreal and Temperate Host Species Growing Near Their Shared Ecotonal Range Limits.Microbial ecology · 2018Article
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- Microbial Community Analysis with Ribosomal Gene Fragments from Shotgun Metagenomes.Applied and environmental microbiology · 2016Article
- Ribosomal DNA and Plastid Markers Used to Sample Fungal and Plant Communities from Wetland Soils Reveals Complementary Biotas.PloS one · 2016Article
- Molecular evidence on the occurrence of co-infection with Pichia guilliermondii and Wuchereria bancrofti in two filarial endemic districts of India.Infectious diseases of poverty · 2014Article
- From genus to phylum: large-subunit and internal transcribed spacer rRNA operon regions show similar classification accuracies influenced by database composition.Applied and environmental microbiology · 2014Article
- Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing.PloS one · 2014Article
- Fungal diversity in permafrost and tallgrass prairie soils under experimental warming conditions.Applied and environmental microbiology · 2013Article
- Fungal community analysis by high-throughput sequencing of amplified markers--a user's guide.The New phytologist · 2013Review
- Improved selection of internal transcribed spacer-specific primers enables quantitative, ultra-high-throughput profiling of fungal communities.Applied and environmental microbiology · 2013Article
- Article
Corrections and comments
- Erratum issued
Authors and funding
2 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project naïve bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50-100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys.
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