Tutorial

Basic Procedures

1. Begin NaPDoS analysis by selecting a domain type
   (KS or C).

2. Select a query type (protein, predicted coding sequence, PCR product, genomic, or metagenomic).

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

• Unless query type protein (amino acid) is specifically selected, sequences should be submitted in nucleic acid format.

• Nucleic acid sequences are translated into predicted amino acids. Translations of genomic and metagenomic data include all 6 possible reading frames.

• Sequences smaller than the minimum size required for reliable domain detection are discarded. (Minimum size for KS domains = 200 aa; C domains = 400 aa). These parameters can be adjusted using Advanced Settings.

• Maximum file size limits are currently set at < 30 MB and < 50,000 individual sequences.

• Please contact us if you would like to analyze larger data sets.

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.

• HMM settings apply to genomic or metagenomic sequence sets only.

• Default C domain HMM parameters are based on recommended trusted cutoff values for PFAM Condensation domain model PF00668.12 [1].

• Default KS domain HMM parameters are based on the Ketosynthase domain model developed by Yadav et al [2].

• Recommended values for minimum protein fragment length and BLASTP e-values were established empirically using manually curated reference database examples.

• For incomplete draft genomes or metagenomes, some candidates detected at this stage may represent partial or overlapping gene fragments or duplications.

• Some candidates identified by HMM search only may encode protein fragments with PKS/NRPS-related functionality (e.g. fatty acid synthases), but do not actually produce compounds traditionally classified as natural products.

• More stringent search methods are applied in later stages of the NaPDoS analysis pipeline to help resolve these issues.

• 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 [3] results for selected candidates and their blast matches in MSF format. The MSF 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.

In some cases, the number of candidate matches on this page may be fewer than the number reported on the earlier genomic summary page, reflecting differences between HMM and BLAST stringencies used for the analysis.

Tree output options

• Phylogenetic domain trees are built using FASTTREE to estimate maximum likelihood [5].

• 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.