Info: 28.6.22

This tutorial is all-deprecated. Use sra-explorer.info to get download links from ENA and Setting up Aspera Connect (ascp) on Linux and macOS to learn how to setup Aspera.

Info: 16.05.2021
The layout of ENA changed so the steps in this tutorial towards finding and selecting datasets are deprecated, and I have not yet found the time to update it. Actually, as there is sra-explorer these days I recommend to just use this to query ENA (or ncbi), see below.

Edit 06.05.20: I actually recommend to use sra-explorer these days to get download links. This is the most convenient and fastest way. It implements the creation of download links from both NCBI and ENA including links for Aspera-driven downloads based on this tutorial. Step 1 of this tutorial can help setting up Aspera. The other steps are redundant if using sra-explorer, but this tutorial has been written before that tool was published. For more details see also sra-explorer : find SRA and FastQ download URLs in a couple of clicks and visit sra-explorer.info. It is self-explanatory.

TUTORIAL

Published sequencing data are commonly stored at NCBI and questions on how to efficiently download these data are posted frequently at Biostars. While NCBI relies on its SRA format to share these data it is possible to directly download them in fastq format from the European Nucleotide Archive (ENA1) which mirrors NCBI.

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.

Step-1: Get the Aspera client

Go to https://www.ibm.com/aspera/connect/ 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:

Linux:

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

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

Mac:

$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

In any case, make sure you add the folder with the ascpexecutable to your PATH. If PATH is a new word to you, please google it ;-)

Step-2: Choose your dataset

We have multiple options here.

Use sra-explorer: The sra-explorer is a convenient GUI to get browse NCBI for datasets. In our case we enter our accession number PRJNA288801 into the search field, then select the desired samples, add them to the cart and then copy the Aspera download links it produces. I will not add further details here as the tool is self-explainatory. It includes many (download) options, check it out. Thanks to Phil Ewels for this awesome tool!

Alternatively, query ENA / NCBI manually to find datasets: 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.

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.

Linux:

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

Mac:

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.

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:

SRRXXXXXX1
SRRXXXXXX2
(...)
SRRXXXXXXn

one can use this simple function to download SRA files via prefetch, followed by fastq conversion with parallel-fastq-dump.

Note In the past prefetch could be coupled with Aspera for fast downloads, this is no longer the case, see https://github.com/ncbi/sra-tools/issues/255. Now prefetch will always download via the standard https connection.

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
  else
    echo '[ERROR]' $1 'apparently not successfully loaded' && exit 1
  fi
}; 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.