The PolyPhen-2 Web interface can be reached at http://genetics.bwh.harvard.edu/pph2/. The input form at this URL allows querying for a single individual amino acid substitution or a coding, non-synonymous SNP annotated in the dbSNP database. After submitting a query, the user is transferred to the Grid Gateway Interface Web page (see GGI User Guide), which is used to track the user’s query progress and retrieve results. Results of the analysis are linked to a separate Web page with more detailed output, formatted as text, graphics, and HTML cross-links to relevant sequence and structural database entries.

An up-to-date Web browser, such as Firefox, Internet Explorer, or Safari. JavaScript support and cookies should be enabled in the browser configuration; the Java browser plug-in is required for the protein 3-D structure viewer to function.

1. Access the PolyPhen-2 Web interface at http://genetics.bwh.harvard.edu/pph2/. In the Protein or SNP identifier text box, enter a protein identifier, e.g., UniProtKB accession number or entry name. For this example, type:

   P41567

   • PolyPhen-2 uses the UniProtKB database as a reference source for all protein sequences and annotations. You can also enter human protein identifiers from other databases (e.g., RefSeq) or standard gene symbols; for the full list of supported databases, see Advanced Configuration Options, “Proteins not in UniProtKB.” However, UniProtKB identifiers are preferred as the most reliable and unambiguous.

2. Leave the Protein sequence in FASTA format text box empty.
3. Enter the position of the substitution in the protein sequence into the Position text box. For this example, type:

   59

4. Select the appropriate boxes for the wild-type (query sequence) amino acid residue AA1 and the substitution residue AA2. For this example, select:

   L
   P

   for AA1 and AA2, respectively.

5. Enter an optional description into the Query description text box:

   Sample query

   • A human-readable query description might help you more easily locate a particular query when you have a large number of pending, running, and completed queries in your PolyPhen-2 Web session.

6. Click the Submit Query button; see Figure 1 for the filled-in query form example. You will be transferred to the Grid Gateway Interface Web page (see Support Protocol 1). Use the Refresh button to reload the page until your query job is listed under Completed.

7. Click the corresponding View link to browse the PolyPhen-2 prediction report for your query; see Figure 2.

   • You will be presented with the Web page listing your Query (mapped to a reference UniProtKB protein if possible) and the Prediction/Confidence panel. Prediction outcome can be one of probably damaging, possibly damaging, or benign. Score is the probability of the substitution being damaging; sensitivity and specificity correspond to prediction confidence (see Background Information, Prediction Algorithm for explanation). The predicted damaging effect is also indicated by a vertical black marker inside a color gradient bar, where green is benign and red is damaging.