Uploading data to the NCBI SRA database

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1 James Madison University From the SelectedWorks of Ray Enke Ph.D. July, 2017 Uploading data to the NCBI SRA database Ray A Enke This work is licensed under a Creative Commons CC_BY-SA International License. Available at:

2 How to cite this work Submitting data to the NCBI SRA Database BIO480/580 Lab James Madison University Dr. Ray Enke This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 United States License. Recommended citation: Enke, R. (2017) Submitting data to the NCBI SRA Database. CSHL DNALC RNA-Seq for the Next Generation Working Group. Objectives Use the Submission Wizard to submit RNA-seq FASTQ files to the NCBI SRA database The NCBI Sequence Read Archive (SRA) database makes biological next generation sequencing (NGS) data freely available to the research community to enhance reproducibility and allow for new discoveries by comparing data sets. The SRA stores raw sequencing data and alignment information from high-throughput sequencing platforms, including but not limited to Illumina sequencers. Total time of protocol: ~1 hour Computing requirements A free NCBI account laptop or desktop computer with a wired internet connection (no wifi) for file transfers FileZilla application download ( Uncompressed (.fastq) or compressed (.fastq.gz) FASTQ files saved locally to your computer or external hard drive Note: The contact sra@ncbi.nlm.nih.gov can be used for questions about your data submission, however, I found this to be a mostly a waste of time. This protocol is meant to serve as a more userfriendly version of the instructions provided on the NCBI SRA website. 1. Uploading FASTQ files to the NCBI server FASTQ files, even when compressed (.fastq.gz) are large and not trivial to move around. The 1st step in this protocol is to gather your FASTQ files somewhere locally (this can be an external hard drive if they are too big to store on your computer) and uploading them to NCBI. There are several upload options that explained in detail on the submission portal help page: This protocol will demonstrate a particular method that 1) does not require the unreliable ASPERA browser plugin, which simply does not work for larger files and 2) does not require command line uploads, which if you re familiar with then you likely do not need this protocol. Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY

3 Create a new folder on your computer or external drive to move/copy your FASTQ files into Give this folder a meaningful name that you will be able to easily find Log into NCBI and navigate to the SRA Submission Portal Wizard: Select the FTP upload option to reveal your FTP connection information You will use this login information in conjunction with a free FTP app called FileZilla to preload your files prior to starting your submission. Download the free FileZilla FTP app ( install and open Plug your FTP login information from above: Host: ftp-private.ncbi.nlm.nih.gov Username: subftp Password: (this is your user-specific password) Select Quickconnect Copy/past Remote site: (this is your user-specific account folder) Hit enter to gain access to your account folder on the NCBI server Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY

4 Create a new directory (folder) in the remote site folder for your submission (right click/control click>new directory) I called my example folder ENKE_FASTQ Once this folder is created, drag your FASTQ files into this folder to begin the transfer This will take a little while; my 12 FASTQ files took ~15 min to transfer Once uploaded, it will take an additional ~10 min for folder to become available Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY

5 2. Creating a New SRA Submission While waiting for your files to transfer, go back to the SRA Submission Portal and start your new submission Select New Submission Enter Submitter information Next enter General Info about your project Create new BioProjects and BioSamples unless your data is part of a previous submission Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY

6 Next enter info about your project If your data is part of the CSHL RNA-Seq for the Next Generation (or other consortium project) enter the description and URL: Link description: RNA-Seq for the Next Generation Project URL: Add pertinent grant funding and/or publications associated with your data on this page Here s the grant info for the CSHL RNA-Seq for the Next Generation project: Infrastructure and Training to Bring Next-generation Sequence (NGS) Analysis Into Undergraduate Education DUE: , National Science Foundation, NSF Select your sample type from a list to generate a sample type-specific attributes template This must be filled out and uploaded as a.tsv text file I found it easiest to download and modify the Excel version then save the finished template as a.tsv text file Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY

7 Here s an example of my attributes file (green=required; yellow=optional) Next download and fill in a sample Metadata spreadsheet This also must be filled out and uploaded as a.tsv text file I also found it easiest to download and modify the Excel version then save the finished template as a.tsv text file There are a few important points here: Keep the bioproject_accession field blank The sample name field must match the sample name from your attributes file Columns E-J + L will all be identical for all members of the CSHL RNA-Seq for the Next Generation group. A GoogleSheet template for this group can be downloaded here: tinyurl.com/yc8krn5x Paired end data files must be entered sequentially into the filename and filename2 columns in the same row for each sample (ie not in separate rows) Here s an example of my metadata file (green=required; yellow=optional) The last step is linking your previously uploaded FASTQ files to your submission Select the I have all files preloaded for this submission option & select your preloaded folder Select the option to continue the rest of the submission automatically Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY

8 FYI, it takes ~10 min after transfer is complete for folder to become available Once the folder becomes available, begin the upload; each file takes a few minutes Submission Portal Wizard Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY

9 After all files are successfully uploaded your submission is complete! You should get a submission confirmation, release date, and overview: You will also get an confirmation: Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY