Tutorial: Fast download of FASTQ files from the European Nucleotide Archive (ENA)
gravatar for ATpoint
8 months ago by
ATpoint14k wrote:

As questions on how to retrieve published sequencing data fast and efficiently are posted here on Biostars quiet frequently, this little tutorial demonstrates how to perform bulk download of fastq files from the European Nucleitode Archive (ENA). Typically people ask on how to get a certain SRA file from NCBI and how to convert it to fastq. The common answer is prefetch followed by fastq-dump, but especially the latter is rather slow, so total file processing might take some time, especially if CPU (and disk) ressources are limited. Luckily, most published (and unrestricted) sequencing data are mirrored at the ENA directly in fastq format, and there is a simple and efficient way to retrieve them. In this tutorial, we will examplarily download an entire dataset of ChIP-seq and ATAC-seq data, requiring minimal preprocessing work. We will use the Aspera client for download rates of several tens of Mb/s up to few hundred Mb/s (depending on the connection, I/O capacity and distance to the download location). This example code should work on Linux and Mac.

--- last modified: 1.3.19 Added link to sra-explorer : find SRA and FastQ download URLs in a couple of clicks

Step-1: Get the Aspera client

Go to https://downloads.asperasoft.com/en/downloads/8?list and get the most recent installer for your system. For Linux, it is a tarball (use tar zxvf to unpack) with an installer batch script and for Mac, a standard disk image.

After installation, there now will be these executables/files in their default locations:


$HOME/.aspera/connect/bin/ascp --- the executable

$HOME/.aspera/connect/etc/asperaweb_id_dsa.openssh --- openssh file that we'll need later


$HOME/Applications/Aspera\ Connect.app/Contents/Resources/ascp --- the executable

$HOME/Applications/Aspera\ Connect.app/Contents/Resources/asperaweb_id_dsa.openssh --- openssh file that we'll need later

Step-2: Choose your dataset

Once you know which data you want to download, check if they are backed up on the ENA, which is true for most unrestricted data. For this tutorial, we will download the entire dataset from the ChIPmentation paper of 2015. When you check the paper for the NCBI accession, you'll find GSE70482. Following this link, you find the BioSample accession number PRJNA288801. So you go to the ENA, enter this PRJNA288801 in the search field and find a summary page with all available data for download. Scrolling down a bit, you see a table with accession numbers and all kinds of metadata. As typically we do not need most of these metadata, we use the field Select columns to select the essential metadata we need for the download, which are Study Accession, FASTQ files (FTP) and Experiment title. After selecting these, and unselecting everything else, you press TEXT and save the file as accessions.txt in your project folder.

Edit: 01/19: Also see sra-explorer : find SRA and FastQ download URLs in a couple of clicks from Phil Ewels for a nice interface to browse data on NCBI and ENA.

Select Columns ENA


Step-3: Download the data

As you'll see in accessions.txt, the download paths direct you to the ENA ftp-server, which is rather slow. We want to download with the Aspera client (up to 200Mb/s at my workplace). Therefore, we awk around a bit to change the download paths to the era-fasp server. As you'll see in case of paired-end data, the paths to the two mate fastq files in accessions.txt are separated by semicolon, which we take into account. The output of this snippet is download.txt.


awk 'FS="\t", OFS="\t" { gsub("ftp.sra.ebi.ac.uk", "era-fasp@fasp.sra.ebi.ac.uk:"); print }' accessions.txt | cut -f3 | awk -F ";" 'OFS="\n" {print $1, $2}' | awk NF | awk 'NR > 1, OFS="\n" {print "ascp -QT -l 300m -P33001 -i $HOME/.aspera/connect/etc/asperaweb_id_dsa.openssh" " " $1 " ."}' > download.txt


awk 'FS="\t", OFS="\t" { gsub("ftp.sra.ebi.ac.uk", "era-fasp@fasp.sra.ebi.ac.uk:"); print }' accessions.txt | cut -f3 | awk -F ";" 'OFS="\n" {print $1, $2}' | awk NF | awk 'NR > 1, OFS="\n" {print "ascp -QT -l 300m -P33001 -i $HOME/Applications/Aspera\ Connect.app/Contents/Resources/asperaweb_id_dsa.openssh" " " $1 " ."}' > download.txt

The output is a simple list of download commands using ascp.

output download.txt

That's it. Now, we only have to run the download commands.

Edit (23.07.18): The download paths are always like era-fasp@fasp.sra.ebi.ac.uk:/vol1(...). I point that out because of a recent post (328182) where OP accidentally forgot the ":" after the .ac.uk and used fasp@ instead of era-fasp@.

Lets download:

## Either by a simple loop:
while read LIST; do
$LIST; done < download.txt

## or by using GNU parallel to have things parallelized:
cat download.txt | parallel "{}"

Once the download is complete, one can play around using the accessions.txt to rename the files with e.g. information from the Experiment title field (column 2), or other metadata you may retrieve from ENA.

Edit 28.2.19: For matters of completeness, I also add a suggestion on how to get the same data from NCBI using prefetch and parallel-fastq-dump, a wrapper for fastq-dump from Renan Valieris for parallelized fastq conversion from sra files. Say one has a file IDs.txt which contains the SRA file IDs like:


one can use this simple function to download SRA files via prefetch (please see the NCBI documention on how use Aspera with prefetch to avoid slow FTP downloads), followed by fastq conversion with parallel-fastq-dump.

function LoadDump {
  prefetch -O ./ -X 999999999 $1 

  if [[ -e ${1}.sra ]]; then
    parallel-fastq-dump -s ${1}.sra -t 8 -O ./ --tmpdir ./ --split-3 --gzip && rm ${1}.sra
    echo '[ERROR]' $1 'apparently not successfully loaded' && exit 1
}; export -f LoadDump

cat IDs.txt | parallel -j 2 "LoadDump {}"

This would use 8 threads for fastq conversion and run two SRA files at a time via GNU parallel, hence requiring 16 threads. As always, scale up or down based on the available resources and potential I/O bottlenecks on your system.

ADD COMMENTlink modified 22 days ago • written 8 months ago by ATpoint14k

Good work! I once had the pleasure to use fastq-dump on whole-genome data. I was cursing in multiple languages! :D

ADD REPLYlink modified 8 months ago • written 8 months ago by Eric Lim1.3k

Good work dude. Will use this next time I need to get data from ENA.

ADD REPLYlink written 8 months ago by Kevin Blighe39k

Thank you Sir, glad to help!

ADD REPLYlink written 8 months ago by ATpoint14k

I've recently started to download FASTQ files via Aspera, but I am using ena-file-downloader.jar. That's is the github link and it is also accessible from "Bulk Download Files" button in the same website. It has GUI, you can choose between FTP and Aspera and you can specify Aspera parameters. Do you think that the speed would be different between the applicaton and the terminal?

ADD REPLYlink written 22 days ago by Batu110

They probably both use the same Aspera server so speed is probably similar. Question would be if this tool you mention allows parallel downloads of several files.

ADD REPLYlink written 22 days ago by ATpoint14k
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