Sequence validation

We check all sequences before running a search. In order to avoid problems with the validation of your sequence, you should use only plain, unformatted text. Here are some of the validation checks that we apply to sequences:

• sequence length must be less than 10,000 residues
• the sequence must be a protein sequence; nucleotide sequences will not be accepted
• only residue symbols allowed in the sequence (letters or "*"); sequences containing other characters will not be accepted.
  Note that "-" was previously accepted as a valid sequence character, but is not allowed in the latest version of HMMER.
• FASTA-header lines are accepted but will be removed

If you have problems getting your sequence to upload, please check that it passes all of these tests. Note that although we do allow FASTA-style header lines on a sequence, some characters in header lines can still cause the sequence to be rejected. If in doubt, please remove header lines before pasting in your sequence.

You can see an example of a sequence that will successfully pass all of the validation tests by clicking the Example button below the search form.

Search options

The default threshold for the HMM search is an E-value of 1.0, but you can also use the gathering threshold for each HMM, or you can specify your own E-value setting. Note that the E-value that you give must be positive.

By default the search will only look for Pfam-A families on your sequence but, by checking the box below, you can also search for Pfam-B hits.

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File contents

We accept only protein sequences and your uploaded file must conform to a fairly strict interpretation of the FASTA file format. We apply the following checks to the format of uploaded sequence files. Files that do not conform to the following rules will be rejected by the server

File contents

Files must contain only header lines and sequence lines. Header lines, which begin with ">", can be used to describe the sequence that follows. There is no fixed format for header lines but we restrict the characters that are allowed. If your header lines contain any of the following characters, your file will be rejected:

; \ ! *

Note that we explicitly include the semi-colon (;) in the list of forbidden characters, although this may be used to denote comments in some versions of the FASTA-format. Please do not use comments in the FASTA files that you upload here.

Header rows

The FASTA-format specification recommends that header lines are kept shorter than 80 characters. Batch searches are run using our pfam_scan.pl script, which uses programs from the HMMER suite. Because of the way that HMMER handles header lines in FASTA files, only the first 60 characters are actually used. Please make sure that your header lines are 60 characters or less in length.

In order to avoid generating strange or ambiguous search results, we require that header lines in your file are unique, at least within the first 60 characters. Uploaded files which contain duplicate header lines will be rejected.

Sequence symbols

Your sequence should be a valid protein sequence. As such, the sequence line should contain only amino-acid symbols, i.e. capital letters excluding "J". In the context of a Pfam search, gaps and translation stops have little meaning and should not normally be used, but we accept "-" or "*" to denote gaps and translation stops respectively.

Service limits

Searches run on a "compute farm" with a limited number of "slots". Each search takes one slot and once all slots are in use, new jobs wait in a queue for the next slot to become free. In order to prevent large jobs occupying slots for very long periods, which can impact the availability of the system for other users, we place a number of restrictions on the size of job that we will accept.

File size

Files must have fewer than 500,000 lines and fewer than 5000 sequences.

Sequence length

Each sequence must be no longer than 20000 amino-acids.

Sequence variation

We use heuristics to check that a sequence has a reasonable level of variation, in order to prevent large strings of identical sequence or a large number of duplicate residues being searched. If you find that you cannot submit a valid sequence because of this restriction, please let us know.

E-value limit

If you specify an E-value cut-off for your search, that E-value must be a positive number.

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The search currently covers the following sections of the database:

• text fields within Pfam entries, such as description and comments
• sequence description and species fields
• the HEADER and TITLE records from PDB files
• Gene ontology (GO) IDs and terms
• InterPro abstracts

You can perform the same search from anywhere within the Pfam site, using the keyword search box at the top right-hand side of every page.

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You can enter either an accession or an identifier for a Pfam family. The search is case insensitive.

We calculate functional similarity using the method described here. We currently apply an arbitrary cut-off of 0.75 to the similarity score, to avoid generating excessive volumes of results.

You can specify one or more domains that must appear in an architecture and one or more domains that must not appear in the result architectures:

1. choose a group of families or the first letter of the family ID from the list below
2. in the left-most list, select the ID of a domain
3. choose Includes or Does NOT include to add the selected domain to the appropriate list
4. continue until the Includes and Does NOT include lists show all of the domains that you need
5. press Submit query to search for architectures matching your specification

Note that the query itself can take some time to run, depending on how many domains you add to each constraint. If the search returns a lot of results, you may also find that your browser hangs momentarily, while the domain graphics are rendered in the page.

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This tool allows you to search DNA sequences of upto 80kb against Pfam HMMs, using the Wise2 package. You can see more information about Wise2 at the homepage.

Because these searches can be time consuming, we run the searches offline and mail the results to you. Please ensure that the email address that you enter is correct, as no checking is performed before the search is run.

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