3. Fetch public RNA-seq data

Today’s we will learn to download FASTQ files from a published paper:

Manuscript: Crotta et al. (2023). Nature Communications. http://doi.org/10.1038/s41467-023-36352-z

STEP 1 : Find where the data is available for download in the above manuscript

Click on the link above and search for “Accession”, “Data availability”, “BioProject ID” or “GEO accession code”
If, only a GEO accession code is available, go to the GEO database and look for BioProject ID - Note, ENA (Step2) requires this identifier to download the data.

Which BioProject ID host the data used in the above manuscript?

Solution

PRJNA862097

STEP 2: Search for data for the identified BioProject ID at the European Nucleotide Archive (ENA) database

Go to https://www.ebi.ac.uk/ena/browser/home and search for the BioProject ID using the search option on the top right corner and click on ‘view’:

STEP3: (if applicable) select one or more BioProject submission(s). Click on the first listed BioProject ID:

STEP4: Select FASTQ files (tick boxes next to the file names) and click on “Get download script”. This will download a bash script (e.g., )

Open the downloaded ena file using TextEdit (NotePad or similar app). The downloaded script looks like this:

wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039511/SRR1039511_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039520/SRR1039520_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039519/SRR1039519_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/004/SRR1039514/SRR1039514_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039521/SRR1039521_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039520/SRR1039520_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039521/SRR1039521_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039510/SRR1039510_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039508/SRR1039508_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039510/SRR1039510_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039518/SRR1039518_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/007/SRR1039517/SRR1039517_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039509/SRR1039509_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/004/SRR1039514/SRR1039514_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039511/SRR1039511_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039519/SRR1039519_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/007/SRR1039517/SRR1039517_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039508/SRR1039508_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039509/SRR1039509_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039518/SRR1039518_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039520/SRR1039520.fastq.gz

Now using the TextEdit or NotePad app, we will add the following lines to the top of the script:

#!/bin/bash -l
#PBS -N ENAdownload
#PBS -l walltime=72:00:00
#PBS -l mem=16gb
#PBS -l ncpus=8

Copy the script to your HPC working folder $HOME/workshop/2024-2/session4_RNAseq/data
See below how to drag and drop the file using File Finder

NOTE: To proceed, you need to be on QUT’s WiFi network or signed via VPN.

To browse the working folder in the HPC type in the file finder:

Windows PC: open file finder and type the address below to connect to your home directory in the HPC. Remember to replace “USER” by your actual user name.

\\hpc-fs\home\USER\workshop\2024-4\session4_RNAseq\data

Mac: open file finder and press “command” + “k” to open prompt, then type the below command. Remember to replace “USER” by your actual user name.

smb://hpc-fs/home/USER/workshop/2024-4/session4_RNAseq/data

Evaluate the nucleotide distributions in the 5'-end and 3'-end of the sequenced reads (Read1 and Read2). Look into the “MultiQC” folder and open the provided HTML report.

Copy the downloaded file to your HPC account or copy the content to a file created in the HPC using Nano (or other text editor)
Add the PBS pro scheduler lines and submit a job. See step by step details at:

eResearch Downloading public data

Source: https://ena-docs.readthedocs.io/en/latest/retrieval/file-download.html#using-ena-file-downloader-command-line-tool

ENA Browser

Go to the ENA Browser https://www.ebi.ac.uk/ena/browser/home

Search NGS data of interest

In the ‘view search box' enter one of the following identifiers:

Project accession (i.e., PRJNA229998)
Study accession (i.e., SRP033351)
Experiment accession (i.e., SRX384360)
Run accession (i.e., SRR1039508)

Once there, you can download any associated files by clicking the relevant links and then clicking on “Get download script”.

For example:

wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039510/SRR1039510_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039510/SRR1039510_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039520/SRR1039520_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039520/SRR1039520_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039511/SRR1039511_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039511/SRR1039511_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039521/SRR1039521_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039521/SRR1039521_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/004/SRR1039514/SRR1039514_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/004/SRR1039514/SRR1039514_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/007/SRR1039517/SRR1039517_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/007/SRR1039517/SRR1039517_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039508/SRR1039508_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039508/SRR1039508_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039518/SRR1039518_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039518/SRR1039518_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039509/SRR1039509_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039509/SRR1039509_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039519/SRR1039519_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039519/SRR1039519_2.fastq.gz

Now create a PBS Pro submission script for the above and save it in a file called, for example ‘launch_ENA_download.pbs’. Note: the below script will download the data in the folder from where the script has been sent to the cluster.

#!/bin/bash -l
#PBS -N download
#PBS -l select=1:ncpus=2:mem=8gb
#PBS -l walltime=24:00:00

#work on current directory (folder)
cd $PBS_O_WORKDIR

wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039510/SRR1039510_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039510/SRR1039510_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039520/SRR1039520_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/000/SRR1039520/SRR1039520_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039511/SRR1039511_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039511/SRR1039511_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039521/SRR1039521_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/001/SRR1039521/SRR1039521_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/004/SRR1039514/SRR1039514_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/004/SRR1039514/SRR1039514_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/007/SRR1039517/SRR1039517_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/007/SRR1039517/SRR1039517_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039508/SRR1039508_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039508/SRR1039508_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039518/SRR1039518_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/008/SRR1039518/SRR1039518_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039509/SRR1039509_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039509/SRR1039509_2.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039519/SRR1039519_1.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR103/009/SRR1039519/SRR1039519_2.fastq.gz

Submit the download script to the cluster:

qsub launch_ENA_download.pbs

Monitor progress of job:

qjobs

Download data using the nf-core/fetchngs pipeline

Source: https://nf-co.re/fetchngs/1.12.0/

Alternatively, to the above approach we can also use the nextflow nf-core/fetchngs pipeline to download data.

To run the this pipeline we need to inputs: 1) list of SRA identifiers and 2) PBS Pro script to fetch the data using sratools.

First, prepare a file with the list of SRA IDs of interest to be downloaded:

Hint:

In the terminal create a new folder called ‘fetchngs’. For example:

mkdir $HOME/workshop/2024-2/session4_RNAseq/data/fetchngs
#then, move to the newly create folder
cd $HOME/workshop/2024-2/session4_RNAseq/data/fetchngs

Copy the following list of IDs. Hint click on the top right corner of the block below to copy the text.

SRR20622172
SRR20622173
SRR20622177
SRR20622176
SRR20622180
SRR20622174
SRR20622178
SRR20622179
SRR20622175

Alternatively, instead of list of SSR identifiers it is possible to download all data in a given BioProject ID:

PRJNA862097

NOTE: Either the list above or citing the BioProject ID in the ‘ids.csv’ file will download exactly the same data.

Create a ‘ids.csv’. file using nano and paste the list of IDs:

nano ids.csv

Next, copy and paste the following PBS script to download the specified files in ‘ids.csv’.
NOTE: instead of listing individual SRR identifiers it is also possible to list the BioProject ID (e.g., PRJNA862107) which will fetch all SRR samples automatically.

Secondly, create a launch PBS script to download the data for the above IDs

Copy the block of code below. Hint click on the top right corner of the block below to copy the text.

#!/bin/bash -l
#PBS -N nf_fetchngs
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=48:00:00
#work on current directory
cd $PBS_O_WORKDIR
#load java and set up memory settings to run nextflow
module load java
export NXF_OPTS='-Xms1g -Xmx4g'
#run the RNAseq pipeline
nextflow run nf-core/fetchngs \
   -profile singularity \
   --input ids.csv \
   --outdir data \
   --download_method sratools \
   --nf_core_pipeline rnaseq \
   -resume

Use nano to create a launch script, for example:

nano launch_nf_core_fetchngs.pbs

Paste the block of code above and save the file.

Submit the the download job to the HPC cluster:

qsub launch_nf_core_fetchngs.pbs

Outputs:

data
├── custom
│   └── user-settings.mkfg
├── fastq
│   ├── SRX16645917_SRR20622180.fastq.gz
│   ├── SRX16645918_SRR20622179.fastq.gz
│   ├── SRX16645919_SRR20622178.fastq.gz
│   ├── SRX16645920_SRR20622177.fastq.gz
│   ├── SRX16645921_SRR20622175.fastq.gz
│   ├── SRX16645922_SRR20622174.fastq.gz
│   ├── SRX16645923_SRR20622173.fastq.gz
│   ├── SRX16645924_SRR20622176.fastq.gz
│   └── SRX16645925_SRR20622172.fastq.gz
├── metadata
│   ├── SRR20622172.runinfo_ftp.tsv
│   ├── SRR20622173.runinfo_ftp.tsv
│   ├── SRR20622174.runinfo_ftp.tsv
│   ├── SRR20622175.runinfo_ftp.tsv
│   ├── SRR20622176.runinfo_ftp.tsv
│   ├── SRR20622177.runinfo_ftp.tsv
│   ├── SRR20622178.runinfo_ftp.tsv
│   ├── SRR20622179.runinfo_ftp.tsv
│   └── SRR20622180.runinfo_ftp.tsv
├── pipeline_info
│   ├── execution_report_2024-08-29_14-23-00.html
│   ├── execution_timeline_2024-08-29_14-23-00.html
│   ├── execution_trace_2024-08-29_14-23-00.txt
│   ├── nf_core_fetchngs_software_mqc_versions.yml
│   ├── params_2024-08-29_14-23-05.json
│   └── pipeline_dag_2024-08-29_14-23-00.html
└── samplesheet
    ├── id_mappings.csv
    ├── multiqc_config.yml
    └── samplesheet.csv