I am trying to identify some wild fungal species using ITS and D1-D2 regions, but in the hits from NCBI getting even different genera in both regions (ITS&D1/D2) for the same species.
Any helpful suggestion ?
Dear Farrukh Baig
Even though NCBI is a great database collecting a lot of information useful for scientists, it has the problem that too many data are registered with wrong species determination, bad sequence quality etc.
Therefore we use some other databases for the molecular idenfication of taxa, which are managed by curators. That means curators ensure the quality of (i) DNA sequences and of (ii) species determination.
First we check databases with curators, then we compare with NCBI...
- UNITE: https://unite.ut.ee/ (go to "run analysis", use field "blastn"). Check the taxon/taxa with the best score bits. Below the list of taxa you will find the nucleotide sequences displayed and they are linked to the corresponding NCBI entry. If your sequence has gaps or differs in some nucleotide, check your raw data. Are you sure your sequence quality is "good"?
- BOLD: http://www.boldsystems.org/index.php/IDS_OpenIdEngine (go to "ITS", use "all barcodes"). You can check the best match on the right column: press on "published". If sequences are depostited in BOLD but still not published, you will not be able to open this file ("early release").
I hope this helps you!
Carolina
Hi
identification of fungi can be solved by following guid books of systematic mycology
which can guide you to genus lvel or sometimes to species level. Now it it is necessary to use specific primer to identify gene of specis .
regards
Farrukh
Carolina is absolutely right about database accuracy. However your question is related with the fact that the ITS region and the LSU region (where D1 and D2 domains are) have two different rates of variation. ITS is considerably variable and LSU is a conserved region. So it is common to have two different results from each region. This is more evident when your taxa has only sequences on GenBank for one of these regions.
If you are trying to identity to the species level, the ITS is more suitable since it has been recognized as the genetic barcode for fungal species. However, the id depends on: the quality of the data in the database, the number of sequences of your taxa in it, and the error probability of the Blast search.
So you have to do a full interpretation of the results of the blast including the similarity, coverage, "e value" (error probability), and to analyze all the top matches not only the first one. If you have a best match with an "e value" near to cero, then you have to interpret if it is reliable by checking its metadata (click in the accession number), this include; if the sequence belong to an already published paper, if it belongs to a taxonomy-systematics paper, and if the autor of that paper has taxonomic experience on that genera.......
Use a taxonomist - a traditional taxonomist, not a molecular biologist
Dear Farrukh Baig
It useful to look out to the morphology and to the microscopy characteristics but not all species can be cultured. Cultures are important for some groups where the modern morphological taxonomy is based entirely on in vitro characters. Molecular technique is important in this case in particular, DNA sequences which is quit useful technique to identify fungal species. The following references could be useful for your question.
Best luck
Keith A. Seifert and Amy Y. Rossman (2010). How to describe a new fungal species. IMA Fungus. 1(2): 109–116.
Hi Farrukh
You can identify the species according to morphology characterization and molecular sequence, but this is general information and need to experience in this field
Molecular tools are a useful way to identify fungi from mycorrhizas, mycelium, cryptic species and anamorphs when scarce or no morphological data were found. But morphology and traditional taxonomy shoud be the first step to identify fungi. Carolina show us some of the problems of the data logged in databases as NCBI. That don't mean molecular tools are useless. I agree fully with Maripaz, molecular tools are useful only when sequences are supported by a morphological analysis made by a taxonomist and then published in the DNA databases.
In general, these methods will get you reliably to the genus level. In most cases, once you know the genus you can look for systematics papers on that genus determine the best way to resolve species. It is also important to make comparisons to "type" material of the species you are interested in.
Unfortunately, UNITE can be next to useless.
For example, only the top 15 alignments are shown. These might all be 'identified' merely as 'Fungi' (because those who submitted the sequences did not know their identity.)
It would be good if the UNITE organisation could delete these 'unknowns'
I agree with Carolina Cornejo and Pablo Pérez Daniëls. In my point a view, in order to identify any microorganism, you need to carry out some tests which their results are confirmatory to each others. These tests are starting from morphological, biochemical and finally molecular identifications. The recent reviews didn't pay attention for biochemical identification.
Farrukh Baig & Mohamed Sheashea
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