Mascot: The trusted reference standard for protein identification by mass spectrometry for 25 years

Sequence database setup: NCBI EST (OBSOLETE)

IMPORTANT – NCBI have dropped gi numbers
In late August 2016, NCBI removed gi numbers from the title lines of the EST Fasta files. This breaks the definitions on this page, so we advise you to switch to the EMBL EST files, which provide essentially the same coverage and have the advantage of being more evenly divided.
If you are part way through a major project or have a workflow that absolutely requires the continued use of gi numbers as identifiers, just freeze your existing EST databases. That is, disable any type of automatic updating. You will not miss much because the rate of increase of the EST divisions of GenBank has dropped to negligible levels.


Three EST databases are compiled by the NCBI (National Center for Biotechnology Information). They contain "single-pass" cDNA sequences, or Expressed Sequence Tags, from the EST divisions of GenBank.

There are currently three EST databases: human, mouse, and others. This document uses the "others" database as an example. To work with the human or mouse databases, simply substitute the word "human" or "mouse" for "others". For example, the human compressed Fasta file is est_human.gz, the keyword is EST_human_from_NCBI, the recommended Mascot name is EST_human, etc.

As of April 2012, the compressed EST_others file from NCBI was 11 GB and the unpacked Fasta was 40 GB. Address space constraints mean that it is no longer possible to memory map this file or to build a taxonomy index using 32-bit executables. Even so, given sufficient disk space, you should still be able to search EST_others on a 32-bit system, provided you configure without memory mapping or taxonomy.

NCBI have no plans to split EST_others, so a more practical alternative is the set of 10 EMBL EST files, which are more evenly divided. As of April 2012, the largest (plants) was 4.3 GB compressed and 17 GB unpacked.

If you only require sequences for a particular organism, another possibility is to download an organism specific EST database.

Download for the latest release.


Taxonomy for NCBI EST databases is predefined in mascot.dat. For EST_others, choose "dbEST FASTA using GI2TAXID". (There is no value in building taxonomy indexes for human or mouse because these are single organism databases.) The following taxonomy files are required:

Note that the taxonomy files go into the taxonomy directory, not into the sequence database directory. Also, some files need to be unpacked (using tar) as well as uncompressed.


NOTE: UniGene was retired by NCBI in July 2019, although the final UniGene builds are still available as static content from the FTP site

The NCBI UniGene indexes are created by automatically partitioning GenBank sequences into non-redundant sets of gene-oriented clusters. If UniGene indexes are available locally, results from Mascot searches of EST databases can be grouped and reported by gene family, rather than by raw EST accession numbers.

In Mascot 2.4, the following UniGene indexes are included in the predefined database definitions. To enable UniGene indexes in earlier versions, refer to your local copy of this help page

  • EST_human
    • Homo_sapiens
  • EST_mouse
    • Mus_musculus
  • EST_others
    • Anopheles_gambiae
    • Arabidopsis_thaliana
    • Bos_taurus
    • Caenorhabditis_elegans
    • Chlamydomonas_reinhardtii
    • Danio_rerio
    • Dictyostelium_discoideum
    • Drosophila_melanogaster
    • Hordeum_vulgare
    • Oryza_sativa
    • Rattus_norvegicus
    • Takifugu_rubripes
    • Triticum_aestivum
    • Xenopus_laevis
    • Zea_mays

Parse Rules

A typical Fasta title line is:

>gi|16764|emb|Z17609.1|Z17609 ATTS0183 Gif-SeedA+B Arabidopsis thaliana cDNA clone YAP043T 3′

The gi number is the most reliable identifier. Suitable parse rules are:

Accession from Fasta title: ">\(gi|[0-9]*\)"
Description from Fasta title: ">[^ ]* \(.*\)"

If an entry in EST_others represents multiple source database entries, the Fasta title lines are concatenated together with CTRL+A as the delimiter.

Configuration (Mascot 2.3 and earlier)

For this example, est_others.gz was downloaded to a folder named C:\Inetpub\MASCOT\sequence\EST_others\current. The file was decompressed using gzip, and renamed to EST_others_20110601.fasta. Taxonomy and memory mapping have been enabled, which is only possible for EST_others when using 64-bit Mascot.

Mascot database maintenance utility

There is no downloadable full text file for EST_others, but full text for individual entries can be retrieved across the web from the NCBI Entrez server. The syntax for the Path field is:


If you don’t require full text in a Mascot Protein View report, simply leave the Host, Port, and Path fields blank and choose
— no full text report —
in the drop down list.

Always test a new definition before applying the changes to mascot.dat.