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

Sequence database setup: NCBI nr

The NCBI nr FASTA file of protein sequences was discontinued in February 2024. We recommend switching to TrEMBL, but you can also download the BLAST nr database and compile the protein sequence database locally; instructions to do so are below.

NCBI gi numbers

In late August 2016, NCBI removed gi numbers from the title lines of the nr Fasta file. Mascot Server has two predefined definitions: NCBIprot, using accession.version identifiers, and NCBInr, using gi numbers.

If you have an old copy of a FASTA file using gi numbers, refer to the old configuration information.

Overview

The nr database is compiled by the NCBI (National Center for Biotechnology Information) as a protein database for BLAST searches. It contains non-identical sequences from GenBank CDS translations, PDB, Swiss-Prot, PIR, and PRF.

The strengths of nr are that it is comprehensive and frequently updated. The downside is that it is a huge database. As of January 2024, the 339 GB Fasta file contained 707 million entries. In most cases, there are better choices of database, such as TrEMBL or a Uniprot complete proteome.

Using the database in Mascot Server 3.0 and later

Follow the instructions for Option 1 or Option 2 below.

Using the database in Mascot Server 2.8

If you are running Mascot Server 2.8.0, 2.8.1 or 2.8.2, please update to 2.8.3. Then follow the instructions for Option 1 or Option 2 below.

Using the database in Mascot Server 2.7 and earlier

Mascot Server 2.7 and earlier are not able to bring the database online due to its sheer size. Please use an alternative database, like TrEMBL, or update to a later version of Mascot Server.

Option 1: Use the 2024 FASTA file

The NCBI nr FASTA file of protein sequences was discontinued in February 2024, but the FASTA file remains available for download. It is simple to enable:

Go to your local Mascot Server home page, Configuration Editor and Database Manager. Under predefined definitions, enable NCBIprot. This will download the 2024 FASTA file.

Option 2: Compile the full BLAST nr database locally

BLAST itself has not been discontinued; only the prebuilt protein FASTA file is finished. It’s still possible to download and compile the BLAST nr database on your local computer. The NCBI announcements point to the BLAST command line instructions. You’ll need to use update_blastdb.pl to download the full nr database. Then you can use blastdbcmd.exe to extract (a subset of) protein sequences in FASTA format.

Downloading BLAST

Download the blast+ package from: https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/. You will also need to install Perl. On Linux, Perl is usually available as part of the operating system. On Windows, Strawberry Perl is a good choice.

Once you have Perl installed, install the blast+ package. It contains the script update_blastdb.pl for initiating the download. On Windows, for example, the script is extracted into the installation directory C:\Program Files\NCBI\blast-2.16.0+\bin: 

  perl "C:\Program Files\NCBI\blast-2.16.0+\bin\update_blastdb.pl" --source ncbi --decompress nr

The command will download the chunked database, like: 

  Downloading https://ftp.ncbi.nlm.nih.gov/blast/db/nr.000.tar.gz...
  Downloading https://ftp.ncbi.nlm.nih.gov/blast/db/nr.001.tar.gz...

Once all the chunks have been downloaded, extract the FASTA formatted database with: 

  "C:\Program Files\NCBI\blast-2.16.0+\bin\blastdbcmd.exe" -entry all -db nr -out nr.fasta

How long will it take? According to the metadata file, in October 2026, the 178 gzip-compressed chunks amount to 369GB. If you have a 1Gbps Internet connection, downloading could theoretically be finished in a couple of hours, but it depends on how fast NCBI’s servers are. With a 100Mbps connection, it will take overnight or longer. In our experience, the chunk downloads sometimes fail so you may need to run the command several times.

In October 2026, the chunks decompress to 709GB. That’s the size of the BLAST database. You will also need disk space for the formatted FASTA file extracted from BLAST, which is, at the time of writing, around 500GB.

  • 370GB for the gzipped chunks
  • Plus 709GB after extracting the files
  • Plus 500GB for nr.fasta

It’s best have at least 2TB of free space. Note that nr.fasta approximately doubles in size every two years!

Enabling the database

Go to your local Mascot Server home page, Configuration Editor and Database Manager.

  1. Choose to create a new database.
  2. Select the option “Use predefined definition template”, and select NCBIprot as the template.
  3. Give it a descriptive name like NCBI_nr_from_BLAST.
  4. Click Next and you have a choice where to put the sequence files. Make sure you choose a directory or drive that has sufficient free space. Twice the size of the FASTA file is sufficient.
  5. Click Create.

Next, go to the Configuration Editor and Configuration Options:

  1. Add NCBI_nr_from_BLAST to the list of databases in IgnoreDupeAccessions and Apply.

At this point, the database definition is waiting for the FASTA file. Don’t use the web browser’s file upload functionality! Web browsers have not been designed to upload a 500GB file over the network. Instead, copy nr.fasta directly to the database’s ‘current’ directory on the Mascot Server hard drive. (On Linux, use sftp or scp or rsync; on Windows, use File Manager or robocopy.) Then, rename nr.fasta to NCBI_nr_from_BLAST_YYYYMMDD.fasta, where YYYYMMDD is today’s date. The database definition should automatically refresh and display the detected FASTA file. Click Enable to start database compression.