| 1. |
- Bengtsson-Palme, Johan, 1985, et al.
(författare)
-
Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data
- 2013
-
Ingår i: Methods in Ecology and Evolution. - 2041-210X. ; 4:10, s. 914-919
-
Tidskriftsartikel (refereegranskat)abstract
- The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and blast searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 – as well as full-length ITS sequences – from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. ITSx paves the way for more sensitive blast searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.
|
|
| 2. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
-
How, not if, is the question mycologists should be asking about DNA-based typification
- 2023
-
Ingår i: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; :96, s. 143-157
-
Tidskriftsartikel (refereegranskat)abstract
- Fungal metabarcoding of substrates such as soil, wood, and water is uncovering an unprecedented number of fungal species that do not seem to produce tangible morphological structures and that defy our best attempts at cultivation, thus falling outside the scope of the International Code of Nomenclature for algae, fungi, and plants. The present study uses the new, ninth release of the species hypotheses of the UNITE database to show that species discovery through environmental sequencing vastly outpaces traditional, Sanger sequencing-based efforts in a strongly increasing trend over the last five years. Our findings chal-lenge the present stance of some in the mycological community - that the current situation is satisfactory and that no change is needed to "the code" - and suggest that we should be discussing not whether to allow DNA-based descriptions (typifications) of species and by extension higher ranks of fungi, but what the precise requirements for such DNA-based typifications should be. We submit a tentative list of such criteria for further discussion. The present authors hope for a revitalized and deepened discussion on DNA-based typification, because to us it seems harmful and counter-productive to intentionally deny the overwhelming majority of extant fungi a formal standing under the International Code of Nomenclature for algae, fungi, and plants.
|
|
| 3. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
-
Improving ITS sequence data for identification of plant pathogenic fungi
- 2014
-
Ingår i: Fungal Diversity. - : Springer Science and Business Media LLC. - 1560-2745 .- 1878-9129. ; 67:1, s. 11-19
-
Tidskriftsartikel (refereegranskat)abstract
- Plant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.
|
|
| 4. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
-
Read quality-based trimming of the distal ends of public fungal DNA sequences is nowhere near satisfactory
- 2017
-
Ingår i: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; 26, s. 13-24
-
Tidskriftsartikel (refereegranskat)abstract
- DNA sequences are increasingly used for taxonomic and functional assessment of environmental communities. In mycology, the nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen marker for such pursuits. Molecular identification is associated with many challenges, one of which is low read quality of the reference sequences used for inference of taxonomic and functional properties of the newly sequenced community (or single taxon). This study investigates whether public fungal ITS sequences are subjected to sufficient trimming in their distal (5’ and 3’) ends prior to deposition in the public repositories. We examined 86 species (and 10,584 sequences) across the fungal tree of life, and we found that on average 13.1% of the sequences were poorly trimmed in one or both of their 5’ and 3’ ends. Deposition of poorly trimmed entries was found to continue through 2016. Poorly trimmed reference sequences add noise and mask biological signal in sequence similarity searches and phylogenetic analyses, and we provide a set of recommendations on how to manage the sequence trimming problem.
|
|
| 5. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
-
Top 50 most wanted fungi
- 2016
-
Ingår i: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; 12, s. 29-40
-
Tidskriftsartikel (refereegranskat)abstract
- Environmental sequencing regularly recovers fungi that cannot be classified to any meaningful taxonomic level beyond “Fungi”. There are several examples where evidence of such lineages has been sitting in public sequence databases for up to ten years before receiving scientific attention and formal recognition. In order to highlight these unidentified lineages for taxonomic scrutiny, a search function is presented that produces updated lists of approximately genus-level clusters of fungal ITS sequences that remain unidentified at the phylum, class, and order levels, respectively. The search function (https://unite.ut.ee/top50.php) is implemented in the UNITE database for molecular identification of fungi, such that the underlying sequences and fungal lineages are open to third-party annotation. We invite researchers to examine these enigmatic fungal lineages in the hope that their taxonomic resolution will not have to wait another ten years or more.
|
|
