Tutorial

Basic Procedures

1. Begin NaPDoS analysis by selecting a domain type (KS or C) and query type (amino acid or nucleic acid).

   • Nucleic acid sequences are translated into predicted amino acids using all 6 possible reading frames.

2. Enter query sequence(s) in fasta format, either by pasting into the text box provided, or uploading a fasta format file.

• Maximum file size limits are currently set at < 500 MB and < 250,000 individual sequences.

• For larger data sets, see our file size management recommendations.

3. Default parameters can be adjusted by clicking on the Advanced Settings link, but caution should be used in interpreting results obtained with modified parameters.

• Less stringent BLAST selection criteria may increase false positives.

• Shorter minimum alignment lengths can decrease sensitivity and classification accuracy, especially for novel domains without exact matches in the reference database.

• Quantitative validation results supporting these caveats are presented below.

4. Clicking on the "seek" button brings up a new window showing estimated processing time and search parameters to be used.

• Analysis does not actually begin until the "submit job" button on this new window is clicked.

• Output selected sequences provides trimmed candidate sequences in a fasta file format. These sequences can then be used to perform BLAST searches against the NCBI database (highly recommended), in case similar domains might not yet have been added to NaPDoS.

• Output alignment will display MUSCLE [1] results for selected candidates and their BLAST matches in MSF, fasta, or clw (Clustal W) or format. The alignment output file can be downloaded for additional offline analysis, for example to make manual adjustments to the alignment, or create custom trees using alternative programs.

• Construct tree progresses to the next stage of the NaPDoS analysis pipeline, inserting candidate sequences plus their BLAST matches into a manually curated alignment of previously characterized database sequences.

Selected candidate sequences plus their BLAST matches are trimmed and inserted into a manually curated amino acid reference alignment, keeping the original reference alignment intact. This alignment is used to build a tree, which is often more useful than BLAST results alone in predicting whether pathway products for candidate domains are likely to be similar or different from previously known examples [2].

Tree output options

• Phylogenetic domain trees are built using FastTree to estimate maximum likelihood [3].

• Tree building does not actually begin until the "submit job" button is clicked.

• After the tree is built, users choose either Newick (plain text) format or an SVG graphics image as an output format.

• User sequences are highlighted in the SVG graphics image format with red dots, as shown in the example below.

• FastTree output does not include bootstrap values. However, the program does provide confidence values, which are included in the Newick format output. These values can be visualized by opening the Newick file with most stand-alone GUI interface tree viewing programs, for example the open source software FigTree.

Typical amino acid percent identities vary widely within different functional domain categories, due at least partly to variability in the number and diversity of reference examples available. The following chart of leave-one-out (jack-knife) cross validation scores, shows averages and expected ranges for members of different functional classes. These values were obtained by finding the closest, non-self database match for each NaPDoS2 reference domain, and comparing amino acid match statistics to other members of the same functional classification category. Brackets after each category on the x-axis indicate the number of database reference sequences analyzed for that category.