I grew up a weird looking fungal colony on SDA at 4C. I isolated it, did a DNA extraction, ran a PCR using fungal primers ITS1F and ITS4. I then sent it for Sanger sequencing. I have the sequence back and the nearest match is to human DNA chromosome 19 at 84%. This has left me somewhat confused! How do I go about putting the sequence into a fungal database and see if I get any hits? Which ones do people use?
Hi Caroline,
In case of bacterial 16S rRNA gene, using the primers is one of the prerequisite criteria to get sequences related to bacteria. For example using primers such as V3-V4 gives you higher percentage of not sequencing other organisms such as eukaryotic cells. For fungal ITS, I don't know if this the same but anyway if it trues you need to know such scenario for your case.
Hope answered your question
Best
Hi,
try this link and let us know.
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=MicrobialGenomes
Thanks, that appears to be for prokaryotes. But I've identified them through UNITE database
did you tryed also to use blastn using the option Organism: fungi (taxid:4751)
It gives you back the results only in such kingdom, and may be more accurate because its frequent update!! Actually is the best way to identify using alignment against a database. then, if you get ambiguous results, you can use alternative taxonomically relevant marker that allow you to disambigue your data. in he link you can find an impressive job done, during which the authors designed universal primers for fungal identification.
Hope it will be usefull,
Regards
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713107/
H165
Hi Caroline
See this paper 1
https://www.ncbi.nlm.nih.gov/pubmed/17228792
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