ENA Bulk RNA-seq Upload Guide

This guide describes the steps required to configure a Linux environment and upload bulk RNA-seq data to the European Nucleotide Archive (ENA). It is based on a real workflow and highlights practical issues and solutions.

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1. Registering Samples and Metadata on ENA

Before uploading any files, you must register your project, study, and samples on ENA.

1.1 Create an ENA Account

• Go to: https://www.ebi.ac.uk/ena/submit/webin/login
• Create a Webin account
• Save your Webin username and password (you will need them later for uploads). Also the username will be given automatically via the registration form.

⚠️ Important: For the next steps, study and sample registration, you can check the ENA documentation

1.2 Register a Study

• Log into the Webin portal

• Clic on Register Study on Study (Project)

• Provide:

  • Release date (when the data will be submitted publicaly) you can change it at any time.
  • Study Name
  • short descriptive study title (what is the difference, I don't know)
  • Abstract

• After submission, you will receive a Study accession (e.g., PRJEBXXXX)

1.3 Register Samples

The best way to register sample is

• Click on Register Samples in Samples
• Download speadsheet to register samples
• Find the best template to you data or go to Other Checklist then ENA default sample checklist.
• Add optional Fields (you can do it later)
• Next
• Download TSV Template.

Then you need to fill the TSV template with the sample information that you want. You can always add a column if needed.

Example Sample TSV

sample_alias	title	tax_id	scientific_name	description
sample_1	Sample 1	9606	Homo sapiens	Control sample
sample_2	Sample 2	9606	Homo sapiens	Treated sample

Notes:

• sample_alias: unique identifier for each sample

⚠️ It will be the id used later to identify your RUN !

• sample_title: This will be the name that you will see in the Webin and not the sample_alias
• tax_id: NCBI taxonomy ID (e.g., 9606 for human or 10090 for mouse)
• scientific_name: must match taxonomy (Mus musculus for example)

Submission

• Upload the TSV via Webin on Register Samples in Samples and Upload filled spreadsheet to register samples. If there is problem in your TSV they will give you an error message.
• After submission, each sample will receive a Sample accession (e.g., ERSXXXX)

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2. Installation of Dependencies

ENA uploads require several tools. Using outdated versions will cause failures, so follow these steps carefully.

2.1 Install Java (Latest Version Required)

System package managers (apt, yum) usually provide outdated versions.

Recommended method:

1. Download from: https://openjdk.org/
2. Choose the Linux tar.gz version
3. Extract it:

tar -xzf openjdk-*.tar.gz

4. You don't necesserly need to export the path of java you just need to know where it's located and you will provide this information later:

path/to/tarFile/jdk-*/bin

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2.2 Download webin-cli

1. Go to: https://github.com/enasequence/webin-cli/releases
2. Download the latest .jar file
3. Again, you just ned to know the path of where you have download the webin-cli-9.0.3.jar

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2.3 Install Ruby (>= 3.1)

Required for aspera-cli.

System package managers often provide older versions, so use rbenv.

Install rbenv

To install rbenv you can follow the doc here.

normally it's really strateforward with

Centos:

sudo yum install rbenv

or Ubuntu

sudo apt install rbenv

Install Ruby

rbenv install 3.1.0
rbenv global 3.1.0

Verify installation

gem -v
ruby -v

If Ruby is not detected correctly, ensure your PATH includes:

export PATH="$HOME/.rbenv/shims:$HOME/.rbenv/bin:$PATH"

For fish shell:

fish_add_path $HOME/.rbenv/shims
fish_add_path $HOME/.rbenv/bin

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2.4 Install Aspera CLI

Used for fast and reliable file transfer (strongly recommended). you can check the doc here

Install

gem install aspera-cli
ascli config transferd install

• The second command installs the FASP transfer engine (ascp)

Configure PATH

You must ensure ascp is accessible, otherwise webin-cli will not use it.

Example:

export PATH="$HOME/.aspera/sdk:$PATH"

Adjust the path depending on your installation location.

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2.5 Final Check

Make sure all tools are available:

java -version
gem -v
ascli --version

If everything works, your environment is ready for ENA uploads.

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3. Upload Process

To be completed.

This section will describe:

• Preparing metadata for runs and experiments
• Using webin-cli for validation and submission
• Uploading FASTQ files with Aspera
• Handling common errors

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Notes and Tips

• Using Aspera (ascp) significantly reduces transfer errors compared to FTP
• Always validate metadata before attempting upload
• Keep track of all accession numbers (Study, Sample, Run)
• Store scripts and commands used for reproducibility