Stockholm format is a multiple sequence alignment format used by Pfam, Rfam and Dfam, to disseminate protein, RNA and DNA sequence alignments. The alignment editors Ralee, Belvu and Jalview support Stockholm format as do the probabilistic database search tools, Infernal and HMMER, and the phylogenetic analysis tool Xrate. Stockholm format files often have the filename extension or.

Syntax

A well-formed stockholm file always contains a header which states the format and version identifier, currently '# STOCKHOLM 1.0'. The header is then followed by a multiple lines, a mix of markup (starting with ) and sequences. Finally, the "//" line indicates the end of the alignment. An example without markup looks like: // Sequences are written one per line. The sequence name is written first, and after any number of whitespaces the sequence is written. Sequence names are typically in the form "name/start-end" or just "name". Sequence letters may include any characters except whitespace. Gaps may be indicated by "" or "". Mark-up lines start with. The "parameters" are separated by whitespace, so an underscore ("_") instead of space should be used for the 1-char-per-column markups. Mark-up types defined include:

Recommended features

These feature names are used by Pfam and Rfam for specific types of annotation. (See the Pfam and the Rfam documentation under "Description of fields")

#=GF

Pfam and Rfam may use the following tags: Compulsory fields: -- AC Accession number: Accession number in form PFxxxxx (Pfam) or RFxxxxx (Rfam). ID Identification: One word name for family. DE Definition: Short description of family. AU Author: Authors of the entry. SE Source of seed: The source suggesting the seed members belong to one family. SS Source of structure: The source (prediction or publication) of the consensus RNA secondary structure used by Rfam. BM Build method: Command line used to generate the model SM Search method: Command line used to perform the search GA Gathering threshold: Search threshold to build the full alignment. TC Trusted Cutoff: Lowest sequence score (and domain score for Pfam) of match in the full alignment. NC Noise Cutoff: Highest sequence score (and domain score for Pfam) of match not in full alignment. TP Type: Type of family -- presently Family, Domain, Motif or Repeat for Pfam. -- a tree with roots Gene, Intron or Cis-reg for Rfam. SQ Sequence: Number of sequences in alignment. Optional fields: DC Database Comment: Comment about database reference. DR Database Reference: Reference to external database. RC Reference Comment: Comment about literature reference. RN Reference Number: Reference Number. RM Reference Medline: Eight digit medline UI number. RT Reference Title: Reference Title. RA Reference Author: Reference Author RL Reference Location: Journal location. PI Previous identifier: Record of all previous ID lines. KW Keywords: Keywords. CC Comment: Comments. NE Pfam accession: Indicates a nested domain. NL Location: Location of nested domains - sequence ID, start and end of insert. WK Wikipedia link: Wikipedia page CL Clan: Clan accession MB Membership: Used for listing Clan membership For embedding trees: NH New Hampshire A tree in New Hampshire eXtended format. TN Tree ID A unique identifier for the next tree. Other: -- FR False discovery Rate: A method used to set the bit score threshold based on the ratio of expected false positives to true positives. Floating point number between 0 and 1. CB Calibration method: Command line used to calibrate the model (Rfam only, release 12.0 and later)

#=GS

Rfam and Pfam may use these features: Feature Description

• ---      AC             ACcession number
  

DE DEscription DR <db>; ; Database Reference OS Organism (species) OC Organism Classification (clade, etc.) LO Look (Color, etc.)

#=GR

Feature Description Markup letters --- --- -- SS Secondary Structure For RNA [.,;<>{}[]AaBb.-_] --supports pseudoknot and further structure markup (see WUSS documentation) For protein [HGIEBTSCX] SA Surface Accessibility [0-9X] (0=0%-10%; ...; 9=90%-100%) TM TransMembrane [Mio] PP Posterior Probability [0-9*] (0=0.00-0.05; 1=0.05-0.15; =0.95-1.00) LI LIgand binding [] AS Active Site [] pAS AS - Pfam predicted [] sAS AS - from SwissProt [*] IN INtron (in or after) [0-2] For RNA tertiary interactions: -- tWW WC/WC in trans For basepairs: [<>AaBb...Zz] For unpaired: [.] cWH WC/Hoogsteen in cis cWS WC/SugarEdge in cis tWS WC/SugarEdge in trans notes: (1) {c,t}{W,H,S}{W,H,S} for general format. (2) cWW is equivalent to SS.

#=GC

The list of valid features includes those shown below, as well as the same features as for #=GR with "_cons" appended, meaning "consensus". Example: "SS_cons". Feature Description Description --- --- -- RF ReFerence annotation Often the consensus RNA or protein sequence is used as a reference Any non-gap character (e.g. x's) can indicate consensus/conserved/match columns .'s or -'s indicate insert columns ~'s indicate unaligned insertions Upper and lower case can be used to discriminate strong and weakly conserved residues respectively MM Model Mask Indicates which columns in an alignment should be masked, such that the emission probabilities for match states corresponding to those columns will be the background distribution.

Recommended placements

Size limits

There are no explicit size limits on any field. However, a simple parser that uses fixed field sizes should work safely on Pfam and Rfam alignments with these limits:

Examples

A simple example of an Rfam alignment (UPSK RNA) with a pseudoknot in Stockholm format is shown below: AF035635.1/619-641 UGAGUUCUCGAUCUCUAAAAUCG M24804.1/82-104 UGAGUUCUCUAUCUCUAAAAUCG J04373.1/6212-6234 UAAGUUCUCGAUCUUUAAAAUCG M24803.1/1-23 UAAGUUCUCGAUCUCUAAAAUCG // Here is a slightly more complex example showing the Pfam CBS domain: O83071/192-246 MTCRAQLIAVPRASSLAEAIACAQKMRVSRVPVYERS O83071/259-312 MQHVSAPVFVFECTRLAYVQHKLRAHSRAVAIVLDEY O31698/18-71 MIEADKVAHVQVGNNLEHALLVLTKTGYTAIPVLDPS O31698/88-139 EVMLTDIPRLHINDPIMKGFGMVINN..GFVCVENDE O31699/88-139 EVMLTDIPRLHINDPIMKGFGMVINN..GFVCVENDE //