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Simplified guidelines to submit 16S rRNA-gene seq data to ENA

ENA (European Nucleotide Archive) is the repository for sequence data hosted by EMBL-EBI (European Molecular Biology Laboratory - European Bioinformatics Institute).

ENA metadata model

Metadata

Data files

What to submit

Ordered list of conceptual steps to undertake for data submission:

  1. study: place-holder for the project
  2. samples: place-holder for the sequence data
  3. raw reads: sequencing information (experiment) + sequence data files (run)

How to submit

Interactive

With this method, you’ll use a mix of the web-browser and the server command line.

  1. First, go to the EBI-ENA website and register a study: from the dashboard, click on Register study. Add a project title and short description + abstract. Download the receipt: remember, you’ll need the project accession number (e.g. PRJEB77087) to link samples and reads to the project/study.

  2. Register samples: download the spreadsheet template \(\rightarrow\) Environmental Checklist \(\rightarrow\) GSC MIxS host associated. Move sample_alias to the first column in the spreadsheet. Add optional columns if relevant: e.g. host taxid (Bos taurus: 9913), host body site (gut), host sex. Remeber to download the accession file after registering the samples: this file will contain unique accession numbers for each sample, that will be needed later to match samples and fastq files.

  3. Fill in the spreadsheet:
    • unique sample_alias and/or sample_title (name) from the mapping file (file of metadata); make sure that this is a unique ID also across experiments (sequence of 1, 2, 3 … N is not a good choice)
    • tax_id (id codes from NCBI);
    • scientific_name, the name associated to tax_id (e.g. bovine gut metagenome);
    • project name, the project accession number from point 1) (see above);
    • collection date, geographic location and coordinates; importantly, you need to fill in also the environmental context and medium (from EnvironmentalOntology): you can repeat the same ontology code for all fields (e.g. UBERON_0001007)

  4. Upload the sample spreadsheet. This step can involve a number of iterations, as the EBI-ENA platform will run a number of check on the consistency of the information provided in the spreadsheet

  5. Upload the fastq files to the EBI-ENA archive: here you’ll need access to the server where your files are stored, or to your local computer provided with a Unix or MAC OS:
    • first, identify the files that you need to upload (e.g. paired-end R1/R2 fastq files for the samples that you want to publish) and copy them to a temporary folder
    • generate md5sum check codes: e.g. for f in *.gz; do md5sum $f | awk '{ gsub(/.*\//,"",$2); print $2"\t" $1 }'; done > md5sums.tsv - move to the temporary folder where the files are, and connect to the EBI FTP server: ftp webin.ebi.ac.uk (you’ll need your webin login details: here)
    • issue the prompt command (from the command line), to disable the interactive mode (otherwise you’ll be asked to confirm the upload of each fastq file)
    • now, upload the file using mput: e.g. mput *.fastq.gz
  6. Now you need to submit your reads: to do so, you have to download and fill the spreadsheet:
    • Download spreadsheet template for Read submission \(\rightarrow\) Submit paired-end reads using two fastq files
    • fill it in with the file names (R1 and R2) and their respective md5 code, with the sample id (from the sample registration), and with additional info (e.g. project accession number etc.) (an R script can be used for this task)
    • upload the fastq files spreadsheet (the ENA platform will run some internal checks for file consistency)

DONE!

Next steps

How can we make this process a bit more automatic? In a next post we’ll look at the programmatic way to upload sequencing files to the EBI-ENA archive.

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