Submitting High-Throughput Sequence Data to GEO (Gene Expression Omnibus)

This tutorial provides a step-by-step guide to submit high-throughput sequence data to GEO. GEO (Gene Expression Omnibus) is a public repository for gene expression data and other functional genomics datasets.

Step 1: Understand Submission Guidelines

1. Visit the GEO Home Page: Navigate to GEO’s home page.
2. Review Submission Guidelines: Go to the submission guidelines at www.ncbi.nlm.nih.gov/geo/info/seq.html.
   • These guidelines provide details on required files and submission procedures.
3. Required Components for Submission:
   • Metadata: Includes study and sample descriptions and protocols.
   • Processed Data: Data used to draw conclusions in your study.
   • Raw Data: These files are deposited into SRA by NCBI staff.

Step 2: Prepare Metadata Spreadsheet

1. Download the Metadata Template:
   • Click on Metadata spreadsheet and then Download metadata spreadsheet on the submission guidelines page.
2. Fill in Metadata:
   • Metadata Tab: Provide detailed information for your study, samples, and protocols.
   • Fields marked with an asterisk (*) are required. Ensure all required fields are completed.
   • Use tips available in the spreadsheet by hovering over the field headers.
3. Review Instructions:
   • Use the Instructions tab in the spreadsheet for guidance.
   • Refer to example worksheets for different experiment types.
4. Special Notes:
   • For paired-end fastq files, list both R1 and R2 files in the same row under the Samples section.
   • Ensure filenames are unique and match submitted files exactly. Avoid whitespace or special characters in filenames.

Step 3: Format Data Files

1. Raw Data:
   • Typically provided in fastq or bam formats.
   • Check SRA’s accepted formats here.
2. Processed Data:
   • Include final quantified data (e.g., normalized read counts for RNA-seq studies).
   • Do not submit only differentially expressed genes or read alignment files as processed data.
3. Organize Files:
   • Place raw and processed data files in a single directory for each experiment.
   • Do not compress fastq or bam files into .tar or .zip archives.
   • Avoid subdirectories with identically named files.

Step 4: Transfer Files to GEO

1. Set Up FTP Transfer:
   • Log in to your My NCBI account.
   • Go to the GEO FTP submission page at geo/info/submissionftp.
   • Click the Transfer Files button to create your personalized upload space.
2. Upload Files:
   • Transfer your folder containing all data files to the FTP upload space.
   • Follow the instructions provided on the GEO FTP page.

Step 5: Upload Metadata File

1. Access Metadata Upload Page:
   • Use the Upload Metadata button to navigate to the metadata submission page.
2. Select FTP Subfolder:
   • Link the uploaded raw and processed data files to the metadata file.
3. Upload Metadata:
   • Select and upload your local metadata file.
   • Specify the release date for public access (maximum four years from the upload date).
   • Provide additional comments if needed in the Comment to GEO staff box.

Step 6: Validate Submission

1. Metadata Validation:
   • After submission, the metadata file is assessed for missing fields or errors.
   • Address any identified issues by uploading a revised metadata file.
2. Receive Confirmation:
   • Upon successful validation, you’ll receive a confirmation message and an email summarizing your submission.

Step 7: Process and Access Records

1. Processing:
   • Your submission enters the GEO processing queue.
   • Upon processing, you’ll receive a GEO accession letter via email with accession numbers.
2. Reviewer Access:
   • The email will also include instructions for creating a reviewer access token.