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PPTdb Tutorial:
Select putative Nucleotide Transporter (NTT) genes from seven Microsporidia species
  • Step 1
  • Step 2
  • Step 3
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  • Step 5
  • Step 6
  • Horizontal gene transfer events are known as evolutionary driving forces of eukaryotes [PMID:24323918]. For example, nucleotide transporter (NTT) gene acquisition was reported as a major evolutionary innovation of Microsporidia which are intracellular parasites of animals and human[PMID:29703975].

    A proof of principle experiment, sequences of putative NTTs were identified from seven Microsporidia species using PPTdb’s iterative functional mining workflow.

    Step 1. Choose your targets.
    Set the parameter, and select interested Species by clicking the left-hand side species-selection panel

    Step 2. Iterative functional mining by enter keyword "nucldotide".
    Comparing to traditional one-way search interface allowing single query once a time. Users must enter an exactly correct term, for example, “nucleotide transport” returns only 1 GO descriptions in A. Algerae PRA339 page. This is because words between “nucleotide” and “transport” cannot be searched by the general query method. In iterative functional mining, one can first search word “nucleotide” which returns all GO descriptions which contains keyword “necltotide”.
    The type-n-search data table of PPTdb allows a secondary refining search by entering “transport” to narrows search results down to 29 GO descriptions. This two-way search interface offers a high flexibility especially on multiple keyword combinations which cannot be searched by one-way search, for example, nucleotide-sugar transmembrane transporter activity (GO:0005338), guanine nucleotide transmembrane transporter activity (GO:0001409), and nucleotide transmembrane transporter activity (GO:0015215). Moreover, the type-n-search table instantly returns database query candidates. Users can be hinted while entering a few characters instead of entire searching keywords, for example, “transport” returns all the candidate entries including “transport”, “transporter”, and “transmembrane transporter” which can be act as a search guidance to inform users that there is more than one “transport” associated keywords in the GO description database.

    Step 3. Set the GO terms as your functional element filter.

    Step 4. Download the FASTA sequences/Annotations/Blast alignment results.
    In this example, 22 putative transporters were identified by those nucleotide transport associated GO descriptions from seven Microsporidia species. Table 3 lists the search results. Interestingly, more than half of the results are hypothetical proteins. The protist uniqueness genes and human transporter homologs can be easily separated from the search. Finally, we can download all the FASTA sequences by clicking the Download link of the web page.

    Step 5.

    FASTA sequences (human homolog), FASTA sequences (protist uniqueness)

    Step 6. Build the phylogenetic tree.
    Tools such as MAFFT [PMID: 28968734] and ClustalW [PMID: 7984417] can do the multiple sequence alignment and deliver the phylogenetic tree of these sequences.