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SRA Toolkit Installation and Configuration Guide

Contact: sra-tools@ncbi.nlm.nih.gov

The following guide will outline the download, installation, and configuration of the SRA Toolkit. Detailed information regarding the usage of individual tools in the SRA Toolkit can be found on the tool-specific documentation pages.

The NCBI SRA Toolkit enables reading ("dumping") of sequencing files from the SRA database and writing ("loading") files into the .sra format (Note that this is not required for submission). The Toolkit source code is provided in the form of the SRA SDK, and may be compiled with GCC. However, pre-built software executables are available for Linux, Windows, and Mac OS X, and we highly recommend using these pre-built executables whenever possible.

Downloading and installing the SRA Toolkit

Download the Toolkit from the SRA website

  1. If you are using a web browser, the following page contains download links to the most current version of the toolkit for each of the supported platforms: SRA Toolkit download page: http://www.ncbi.nlm.nih.gov/Traces/sra/?view=software
  2. If you are instead working from a command line interface, you may use FTP or wget to obtain the software from the following directory: "http://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/current". Example:
    wget "http://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/current/sratoolkit.current-centos_linux64.tar.gz"

Unpack the Toolkit:

  1. For Linux, use tar:
    tar -xzf sratoolkit.current-centos_linux64.tar.gz
  2. For Mac OS X, double-click on the .tar.gz file and the Archive Utility will unpack it. Alternatively, command-line tar will also work (see Linux example, above).
  3. For Windows, either use an archiving and compression utility (e.g., Winzip, 7-Zip, etc.), or simply double-click on the .zip file and drag the 'sratoolkit...' folder to the preferred install location.

Note: For most users, the Toolkit functions (fastq-dump, sam-dump, etc.) will not be located in their PATH environmental variable. This may require providing directory information about the location of the Toolkit. See the below examples for how 'fastq-dump' would be called in different circumstances:

  • ~/[user_name]/sra-toolkit/fastq-dump
    YES: The Toolkit "bin" directory has been placed in the user-specified directory "sra-toolkit"
  • ./fastq-dump
    YES: The Toolkit components are the in the current working directory
  • fastq-dump
    NO: If the toolkit location is not specified in your $PATH variable, then the OS cannot locate the fastq-dump program, even if it is in the current directory. NOTE: Windows users should be able to enter only "fastq-dump.exe" if you have navigated to the Toolkit "bin" directory.

Testing the Toolkit configuration

The Toolkit comes with a default configuration that will work for most users. You may elect to perform the following tests to confirm that your configuration is working correctly. The default location for the "download repository" is:

  • Linux: /home/[user_name]/ncbi/public
  • Mac OS X: /Users/[user_name]/ncbi/public
  • Windows: C:\Users\[user_name]\ncbi\public

Note that if the tests fail, or if you wish to specify the download location for files sourced from NCBI, you should configure your Toolkit installation. During normal operation, the Toolkit may be required to download the following types of data to the default location:

  • Reference sequences: Small (most less than 70 MB) sequences used to decompress aligned SRA data.
  • SRA data files: If data are downloaded "on-demand" using the toolkit, then partial and whole SRA datasets (most are several Gb in size) can be located here. Note: Manually downloaded SRA data obtained using a web browser, wget, ascp, or FTP may be stored anywhere in the local file system.

For the test, we are using an arbitrary dataset, SRR390728 (RNA-Seq (polyA+) analysis of DLBCL cell line HS0798), from the National Cancer Institute’s Cancer Genome Characterization Initiative (CGCI) Project. It is a reasonably small SRA dataset that contains aligned (reference-compressed) data, allowing us to test multiple aspects of the toolkit simultaneously.

  1. Open a terminal or command prompt and "cd" into the directory containing the toolkit executables (e.g., [download_location]/sratoolkit[version]/bin/).
    • Linux and OS X users should execute the following command:
      ./fastq-dump -X 5 -Z SRR390728
    • Windows users should execute the following command:
      fastq-dump.exe -X 5 -Z SRR390728
  2. If successful, the test should connect to NCBI, download a small amount of data from SRR390728 and the reference sequence needed to extract the data, and stream the first 5 spots of the file ("-X 5" option) to the screen ("-Z" option).
  3. If the configuration is not valid, an error like the following will likely be displayed:
    fastq-dump.2.x err: item not found while constructing within virtual database module - the path 'SRR390728' cannot be opened as database or table"
  4. If you receive an error like the one above, please configure the toolkit (described in the next section). If you have already configured the toolkit but are still unable to complete the test successfully, please email sra-tools@ncbi.nlm.nih.gov with a full description of steps taken and error messages received.

Configuring the Toolkit

If you are using SRA Toolkit version 2.4 or higher, you should run the configuration tool, located within the bin subdirectory of the Toolkit package.

Go to the "bin" subdirectory for the Toolkit and run the following command:

./vdb-config -i

This tool will setup your download/cache area for downloaded files and references.

A window will open and present the screen below.

The settings displayed here have the default values. Of primary importance is the Workspace Location, which by default is in the ncbi directory within your home directory.

If you have enabled remote access (enabled by default), the toolkit will contact NCBI on demand over HTTP to retrieve the files it needs to complete your commands.

In order for your commands to complete successfully, the toolkit needs sufficient free space. Genomics datasets are quite large; you may need 100's of GB of free space. This is the primary concern when choosing the Workspace Location. Do you have enough free space there for what you intend to do?

If you need to change the Workspace Location, use the tab key to move the cursor (shown red here) to the change button and press space or enter.

This will bring up the file navigation dialog (see below).

if you already know the path to the directory, you may use the Goto button to directly enter that path. Once you have entered or navigated to the correct directory, press tab to get to the OK button to return to the previous screen.

Once you are happy with the settings, use the tab key to get to the Save button and press enter or space.

Press enter or space one more time, then tab to the Exit button and press enter or space. You will then be returned to your shell command prompt.

Links and help documents

Toolkit download: http://www.ncbi.nlm.nih.gov/Traces/sra/?view=software

Toolkit documentation: http://www.ncbi.nlm.nih.gov/Traces/sra/?view=toolkit_doc

Protected Data Usage guide: http://www.ncbi.nlm.nih.gov/Traces/sra/?view=toolkit_doc&f=dbgap_use