Hello Every one,
I have isolated and sequenced ITS regions for identification of fungi by Sangers method. To construct a phylogenetic tree which is the best tool available in on-line databases.
Thank you all
You can get the ITS datasets you want from UNITE (https://unite.ut.ee/).
Some general ITS-related observations are found here:
https://www.researchgate.net/publication/235931750_Five_simple_guidelines_for_establishing_basic_authenticity_and_reliability_of_newly_generated_fungal_ITS_sequences
https://www.researchgate.net/publication/235601668_Incorporating_molecular_data_in_fungal_systematics_a_guide_for_aspiringresearchers
https://www.researchgate.net/publication/255483379_Towards_a_unified_paradigm_for_sequence-based_identification_of_Fungi
Keep in mind that you normally cannot align the fungal ITS region above the, say, genus or subfamily level.
Article Five simple guidelines for establishing basic authenticity a...
Article Incorporating molecular data in fungal systematics: A guide ...
Article Towards a unified paradigm for sequence-based identification of Fungi
You can get the ITS datasets you want from UNITE (https://unite.ut.ee/).
Some general ITS-related observations are found here:
https://www.researchgate.net/publication/235931750_Five_simple_guidelines_for_establishing_basic_authenticity_and_reliability_of_newly_generated_fungal_ITS_sequences
https://www.researchgate.net/publication/235601668_Incorporating_molecular_data_in_fungal_systematics_a_guide_for_aspiringresearchers
https://www.researchgate.net/publication/255483379_Towards_a_unified_paradigm_for_sequence-based_identification_of_Fungi
Keep in mind that you normally cannot align the fungal ITS region above the, say, genus or subfamily level.
Article Five simple guidelines for establishing basic authenticity a...
Article Incorporating molecular data in fungal systematics: A guide ...
Article Towards a unified paradigm for sequence-based identification of Fungi
IDENTIFICATION: I found that in the case of Trichophyton UNITE needs to be revised. There is much confusion within T. mentagrophytes sensu lato. I conclude that one should pick sequences for comparison by hands using taxonomic literature when species-level resolution is required. But in most cases (in my experience - clinically important Candida, Fusarium, Trichophyton, Gymnascella spp.) alignment with CBS sequences gives acceptable result. http://www.cbs.knaw.nl/Collections/BioloMICSSequences.aspx (FUNCBS)
TREE CONSTRUCTION: There is no simple way to construct good tree. Some special knowledge is required. My favourite tool for sequence alignment and tree construction, though not-online, is SeaView http://doua.prabi.fr/software/seaview.
>I found that in the case of Trichophyton UNITE needs to be revised. There is
>much confusion within T. mentagrophytes sensu lato.
That is why UNITE offers third-party web-based sequence annotation, so that anyone with Internet access and expert knowledge of particular lineages of fungi can make such changes. Including, as you mention, highlighting particularly useful and high-quality sequences.
You can also use EzFungi and download the matches you get and do the tree with them.
http://www.ezbiocloud.net/ezfungi
Hi, I am working with the IST1 region in fungi. Because I am doing NGS I have phenomenal amounts of fasta sequences, so using Muscle or EZfungi might take significant amount of time to align... Or, is there a way now (your post is a year old) to generate phylogenetic trees for these amplicons? I'd like more details about the way Rolf Henrik Nilsson and Vaishali Waman suggested please...
Thanks a lot!
The fungal ITS1 region is so variable that you will not be able to compute a scientifically meaningful alignment from amplicon-style NGS data, at least if your data comprise assorted fungi. It us usually not possible to compute meaningful ITS alignments much above the genus level (and sometimes not even that: https://www.researchgate.net/publication/5570465_Mining_metadata_from_unidentified_ITS_sequences_in_GenBank).
If what you have are amplicon-style ITS NGS data of random fungi, then I'm afraid joint phylogenetic analysis seems a closed route to me.
Article Mining metadata from unidentified ITS sequences in GenBank
Ok. Thanks for the kind advice and quick reply despite the fact that this is not what I waned to hear (ha..! science!).
Well, processing the ITS in NGS it might be a bit different than any other markers depending on what are you trying to do. In any case, with Mothur you don't need an alignment, but a reference file with the sequences and other with the taxonomy.
You can use the UNITE ITS database already formatted for that:
http://www.mothur.org/wiki/UNITE_ITS_database
You should probably start looking into the Mothur forum as it has been a quite recurring question for years.
PS: you may as well use QIIME, but I don't know if they have something more defined, i.e. step-by-step protocol
You can use megaX software for the allignment and building of phylogenetic tree of your choice by choosing the organism
Golla Ramanjaneyulu sir, have you solved your problem ? i am also facing the same problem for the construction of Phylogenetic tree for fungus isolates having ITS regions, can you share your experiences and guidance.
Thanks
>i am also facing the same problem for the construction of Phylogenetic tree for
>fungus isolates having ITS regions, can you share your experiences and
>guidance.
Some observations are offered in Article Incorporating molecular data in fungal systematics: A guide ...
In MAFFT you can do the alignment and a basic tree on line https://mafft.cbrc.jp/alignment/server/
For NGS can use DADA2+UNITE
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