Uploading sequence data to ENA

For each project, it is important to also upload the FASTQ files and other raw sequncing files to ENA or SRA (See: sra.md) as soon as the sequencing data arrives. If the study ID already exists, you can create a new sample ID link to the existing study ID. If there is no previous study ID, you can create a new study ID and add all relevant data. See below for more instructions.

Steps to Upload Data to ENA

  1. Create a New Study:

    • Log in to the ENA Webin submission system. The user id is Webin-60428. Ask the system administrator for the password.
    • Click on the "Submit Data" tab.
    • Click on "Create a new study."
    • Fill in the required fields (e.g., study title, abstract, etc.).
    • Click "Save."

  2. Batch Upload Samples:

    • Click on the "Samples" tab.
    • Click on "Download spreadsheet to register samples."
    • Select the spreadsheet for plant samples.
    • Fill in the required metadata fields for each sample.
    • Save the spreadsheet.

  3. Upload Samples:

    • Click on "Upload filled spreadsheet to register samples."
    • Select the spreadsheet you filled out.
    • Click "Upload."

  4. Upload Sequencing Files using CLI:

    • Use the ENA Webin CLI tool to upload sequencing files.
    • The script upload_ftp.sh is the main script, use it like so: sh ./upload_ftp.sh $HOST $USER $PASS $FILE $DIR Where $HOST is the host address (starting with ftp), $USER is the username, $PASS the password, $FILE the file you are uploading, and dir in which directory of the ftp server you want to upload (use ./ if you want to upload to the base directory).

  5. Embargo Data:

    • If needed, set an embargo date for the data.
    • Click "Save."

  6. Check Submission Status:

    • Monitor the submission status in the ENA Webin submission system.
    • Once the submission is complete, you will receive a confirmation email.

Important Metadata for DNA and RNA Samples

For plant ecology and evolution studies, providing detailed metadata is crucial. Below are recommended metadata fields:

Basic Metadata (For Both DNA and RNA)

  • Sample ID: Unique identifier for each sample.
  • Collection Date: Date when the sample was collected.
  • Collection Location: GPS coordinates (latitude and longitude) and a brief description.
  • Collected By: Name of the person(s) who collected the sample.
  • Species Information: Scientific name, common name, and additional taxonomic details.
  • Subspecies Information: Information on subspecies, variety.
  • Population Information: Details about the population from which the sample originates.
  • Subspecific Genetic Lineage Rank & Name: Further details on infraspecific classification (e.g., ecotype, cultivar).
  • Altitude: Altitude of the collection site (in meters).
  • Geographic Location (Region and Locality): Detailed geographic origin of the sample.
  • Tissue Type: Type of tissue sampled (e.g., leaf, root, stem).
  • Developmental Stage: Growth or developmental stage of the organism.
  • Storage Conditions: How and where the sample was stored (e.g., temperature, preservation method).
  • Extraction Method: Protocol or kit used for DNA/RNA extraction.
  • Ploidy: Ploidy level of the genome (e.g., diploid, triploid, tetraploid).
  • Library Preparation: Details on how the sequencing library was prepared (including kit information).

Additional Metadata (From the ENA Plant Sample Checklist)

  • Propagation: Type of reproduction from the parent stock (e.g., sexual/asexual).
  • Soil Classification (FAO): Soil type based on the FAO World Reference Database for Soil Resources.
  • Soil pH: pH measurement of the soil at the collection site (e.g., 6.2).
  • Growth Facility: Type of facility where the plant was grown.
  • Sample Health State: Overall health status of the subject at the time of collection.
  • Sample Disease Status: List any diseases diagnosed at the time of collection.
  • Estimated Genome Size: Estimated size of the genome (in base pairs).
  • Source Material Identifiers: Unique identifiers assigned to the material sample for traceability, Check the Sample collection list of ParisodLab.

When available, include the following additional fields to capture detailed environmental, collection, and treatment information:

  • Observed Biotic Relationship: Describe interactions between the subject organism and other organisms (e.g., parasite on species X; mutualist with species Y).
  • Sample Collection Method: The method employed for collecting the sample (can include a PMID, DOI, URL, or descriptive text).
  • Sample Storage Temperature: Temperature at which the sample was stored (e.g., -80°C).
  • Sample Storage Location: Name of the freezer or storage room.
  • Soil Taxonomic / Local Classification: Local soil classification details.
  • Soil Taxonomic / Local Classification Method: Reference or method used for local soil classification.
  • Soil Type & Method: Soil classification using accepted terms (e.g., from ENVO) and the method used.
  • Soil Texture Measurement & Method: Proportions of sand, silt, and clay (e.g., 40% sand, 30% silt, 30% clay) along with the method used.
  • Sampled Age: Age of the subject at the time of collection.
  • Sample Disease Stage: Stage of disease at the time of collection (if applicable).
  • Broad-scale Environmental Context: Major environmental system of the sample site (e.g., desert, rainforest).
  • Local Environmental Context: Immediate environmental conditions or influencing entities.
  • Amount or Size of Sample Collected: Total volume, mass, or area of the sample.
  • Sampling Time Point: Date/time of collection (ISO 8601 format recommended).
  • Biological Status: Nature of the sample (e.g., wild, natural, inbred, hybrid).
  • Growth Habit: General growth form or habit of the plant.
  • Climate, Gaseous, & Seasonal Environment: Details on the climate, gas concentrations, and season at the time of sampling.
  • Source Material Description: Additional context clarifying the nature of the source material.
  • Organism Phenotype: Key phenotypic traits of the organism.
  • Sample Material Processing: Any processing applied to the sample after collection.
  • Treatment Regimens:
  • Treatment Type: Type of treatment applied to the sample. - Treatment Duration: Duration of the treatment. - Treatment Concentration: Concentration of the treatment. - Treatment Timing: Timing of the treatment. - Treatment Description: Detailed description of the treatment.

Sequencing Library Metadata (Mandatory Fields)

For ENA/NCBI submissions, the following sequencing and library metadata fields are mandatory:

  • sample: Unique identifier for the sample.
  • study: Identifier for the study.
  • instrument_model: Model of the instrument used for sequencing (permitted values as specified by ENA).
  • library_name: Name of the sequencing library.
  • library_source: Source of the library (permitted values).
  • library_selection: Method used for library selection (permitted values).
  • library_strategy: Strategy used for library preparation (permitted values).
  • library_layout: Layout of the library (permitted values).
  • file_name: File name for reads.
  • file_md5: MD5 checksum for the file.

Additional Library Metadata (Optional)

  • library_design: Details regarding the library design.
  • library_construction_protocol: Protocol used for library construction.
  • design_description: Description of the experimental design.
  • insert_size: Insert size of the library.