Read quality-based trimming of the distal ends of public fungal DNA sequences is nowhere near satisfactory

Nilsson, R. Henrik; Sánchez-García, Marisol; Ryberg, Martin; Abarenkov, Kessy; Wurzbacher, Christian; Kristiansson, Erik

doi:10.3897/mycokeys.26.14591

Cited by 10 publications

(10 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…BLAST identity considers individual indels as mismatches and hence results in lower similarity values than the other two approaches for a given sequence pair. It is also more sensitive to homopolymer-based sequencing errors in the query reads and affected by improper trimming of low-quality terminal portions of reference sequences (Nilsson et al 2017 ). As a result, sequences retrieved as best hits in BLAST searches are not necessarily most closely related (e.g.…”

Section: Pairwise Similarity Assessments: Limitations and Solutionsmentioning

confidence: 99%

Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

et al. 2020

View full text Add to dashboard Cite

True fungi (Fungi) and fungus-like organisms (e.g. Mycetozoa, Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.

show abstract

Section: Pairwise Similarity Assessments: Limitations and Solutionsmentioning

confidence: 99%

Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

et al. 2020

View full text Add to dashboard Cite

show abstract

“…A major concern is the reliability of the DNA sequence data ( Bridge et al 2003 , Nilsson et al 2006 ). PCR or cloning errors (including the introduction of chimeras), DNA degradation, and post-processing of chromatograms, have been shown to be a source of sequence variation in at least some groups ( Haas et al 2011 , Sandoval-Sierra et al 2014 , Hughes et al 2015 , Strid et al 2015 , Aas et al 2017 , Nilsson et al 2017 , Thielecke et al 2017 , Bieker & Martin 2018 ). Such DNA sequences are not real and cannot be checked or corrected without access to a physical specimen or, as a minimum, access to the raw sequence reads ( Tripp & Lendemer 2014 ).…”

Section: Reliability and Extent Of Datamentioning

confidence: 99%

Considerations and consequences of allowing DNA sequence data as types of fungal taxa

et al. 2018

View full text Add to dashboard Cite

Nomenclatural type definitions are one of the most important concepts in biological nomenclature. Being physical objects that can be re-studied by other researchers, types permanently link taxonomy (an artificial agreement to classify biological diversity) with nomenclature (an artificial agreement to name biological diversity). Two proposals to amend the International Code of Nomenclature for algae, fungi, and plants (ICN), allowing DNA sequences alone (of any region and extent) to serve as types of taxon names for voucherless fungi (mainly putative taxa from environmental DNA sequences), have been submitted to be voted on at the 11th International Mycological Congress (Puerto Rico, July 2018). We consider various genetic processes affecting the distribution of alleles among taxa and find that alleles may not consistently and uniquely represent the species within which they are contained. Should the proposals be accepted, the meaning of nomenclatural types would change in a fundamental way from physical objects as sources of data to the data themselves. Such changes are conducive to irreproducible science, the potential typification on artefactual data, and massive creation of names with low information content, ultimately causing nomenclatural instability and unnecessary work for future researchers that would stall future explorations of fungal diversity. We conclude that the acceptance of DNA sequences alone as types of names of taxa, under the terms used in the current proposals, is unnecessary and would not solve the problem of naming putative taxa known only from DNA sequences in a scientifically defensible way. As an alternative, we highlight the use of formulas for naming putative taxa (candidate taxa) that do not require any modification of the ICN.

show abstract

“…The generated data should be deposited to sequence repositories just as carefully and as richly annotated as in the case of Sanger sequences to avoid errors derived from the experimental procedure (cf. Nilsson et al, ). The sequencing error rate, determined through the use of known high‐quality reference data, should always be included in the submission, included either in the sequence header or as additional data.…”

Section: Discussionmentioning

confidence: 99%

Introducing ribosomal tandem repeat barcoding for fungi

Wurzbacher

Larsson

Bengtsson‐Palme

et al. 2018

Molecular Ecology Resources

Self Cite

View full text Add to dashboard Cite

Sequence comparison and analysis of the various ribosomal genetic markers are the dominant molecular methods for identification and description of fungi. However, new environmental fungal lineages known only from DNA data reveal significant gaps in our sampling of the fungal kingdom in terms of both taxonomy and marker coverage in the reference sequence databases. To facilitate the integration of reference data from all of the ribosomal markers, we present three sets of general primers that allow for amplification of the complete ribosomal operon from the ribosomal tandem repeats. The primers cover all ribosomal markers: ETS, SSU, ITS1, 5.8S, ITS2, LSU and IGS. We coupled these primers successfully with third-generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens (Basidiomycota), aquatic chytrids (Chytridiomycota) and a poorly understood lineage of early diverging fungi (Nephridiophagidae). In particular, we were able to generate high-quality reference data with Nanopore sequencing in a high-throughput manner, showing that the generation of reference data can be achieved on a regular desktop computer without the involvement of any large-scale sequencing facility. The quality of the Nanopore generated sequences was 99.85%, which is comparable with the 99.78% accuracy described for Sanger sequencing. With this work, we hope to stimulate the generation of a new comprehensive standard of ribosomal reference data with the ultimate aim to close the huge gaps in our reference datasets. K E Y W O R D S discussions on the implementation of long-read sequencing. The authors would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure, NGI and Uppmax for providing assistance in massive parallel sequencing and computational infrastructure. CW and RHN gratefully acknowledge financial support from Stiftelsen Olle Engkvist Byggmästare, Stiftelsen Lars Hiertas Minne, Kapten Carl Stenholms Donationsfond and Birgit och Birger Wålhströms Minnesfond.

show abstract

Read quality-based trimming of the distal ends of public fungal DNA sequences is nowhere near satisfactory

Cited by 10 publications

References 34 publications

Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

Considerations and consequences of allowing DNA sequence data as types of fungal taxa

Introducing ribosomal tandem repeat barcoding for fungi

Contact Info

Product

Resources

About