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miRBase and MirGeneDB

Download Reference microRNA data from miRBase

First, let’s create a folder to store the reference datasets:

mkdir -p $HOME/workshop/2024-2/session6_smallRNAseq/data/references

Now move to the reference folder and download the miRBase datasets using wget in an Interactive session or (see below) use a PBS Pro script.

OPTION #1: Use interactive session to run the following commands:

Fetch microRNA mature sequences:

wget https://mirbase.org/download/mature.fa

Fetch hairpin sequences:

wget https://mirbase.org/download/hairpin.fa

Fetch the genomic coordinated for precursors and mature sequences:

wget https://mirbase.org/download/hsa.gff3

OPTION #2: submit the following PBS Pro script to the cluster. Let’s 1) copy the script to download miRBase files; 2) move to the reference folder; and 3) print the content of the launch_download_miRBase.pbs script:

cp /work/training/2024/smallRNAseq/scripts/launch_download_miRBase.pbs $HOME/workshop/2024-2/session6_smallRNAseq/data/references
cd $HOME/workshop/2024-2/session6_smallRNAseq/data/references 
cat launch_download_miRBase.pbs
#!/bin/bash -l
#PBS -N download_miRBase
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=2:00:00

cd $PBS_O_WORKDIR

wget https://www.mirbase.org/download/hairpin.fa
wget https://www.mirbase.org/download/mature.fa
wget https://www.mirbase.org/download/hsa.gff3

submit the script to the HPC cluster:

qjobs launch_download_miRBase.pbs

monitor progress of job:

qjobs

Fetch public small RNA-seq data

Today we will download small RNA-seq data from the ENA (European Nucleotide Archive).

Manuscript: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1004188

image-20241025-035010.png

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”, “GEO accession code” or “Array Express” identifier.

  • If, only an Array Express accession code is available, then go to https://www.ebi.ac.uk/biostudies/arrayexpress and search for the Array Express identifier. Browse the database to located the identifier for ENA.

  • Hint: it will take a couple of clicks to open multiple pages to find the identifier for the data deposited in ENA.

image-20241025-041940.png

Which is the Array express identifier noted in the above manuscript and to which ENA identifier it relates to?

 Solution

Array Express: E-MTAB-2206 , and ENA identifier: ERP004592

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

STEP3: Select FASTQ files (tick boxes next to the file names) and click on “Get download script”. NOTE: the Script Name will be different for each person downloading the bash script (e.g., )

image-20241025-042425.png

  • STEP 4: Download the metadata information for the study in TSV (Tab-Separated Values) format:

image-20241025-043327.png

Open the file using an app for Text files (e.g., TextEdit, NotePad, etc):

run_accession	sample_accession	experiment_accession	study_accession	tax_id	scientific_name	fastq_ftp	submitted_ftp	sra_ftp	bam_ftp
ERR409882       SAMEA2300497    ERX376249       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409882/ERR409882.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409882/C_31.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409882
ERR409892       SAMEA2300502    ERX376254       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409892/ERR409892.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409892/H_13.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409892     
ERR409893       SAMEA2300504    ERX376256       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409893/ERR409893.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409893/C_36.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409893     
ERR409895       SAMEA2300492    ERX376244       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409895/ERR409895.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409895/H_09.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409895     
ERR409897       SAMEA2300498    ERX376250       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409897/ERR409897.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409897/H_05.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409897
ERR409898       SAMEA2300501    ERX376253       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409898/ERR409898.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409898/C_29.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409898     
ERR409899       SAMEA2300490    ERX376242       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409899/ERR409899.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409899/H_07.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409899     
ERR409879       SAMEA2300495    ERX376247       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409879/ERR409879.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409879/C_39.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409879     
ERR409880       SAMEA2300488    ERX376240       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409880/ERR409880.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409880/H_08.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409880
ERR409883       SAMEA2300487    ERX376239       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409883/ERR409883.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409883/C_35.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409883     
ERR409884       SAMEA2300491    ERX376243       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409884/ERR409884.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409884/H_12.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409884     
ERR409886       SAMEA2300493    ERX376245       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409886/ERR409886.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409886/H_06.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409886     
ERR409888       SAMEA2300503    ERX376255       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409888/ERR409888.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409888/C_38.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409888     
ERR409878       SAMEA2300496    ERX376248       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409878/ERR409878.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409878/C_33.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409878
ERR409889       SAMEA2300500    ERX376252       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409889/ERR409889.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409889/H_03.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409889     
ERR409881       SAMEA2300509    ERX376261       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409881/ERR409881.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409881/H_10.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409881     
ERR409885       SAMEA2300505    ERX376257       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409885/ERR409885.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409885/H_02.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409885     
ERR409894       SAMEA2300507    ERX376259       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409894/ERR409894.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409894/H_14.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409894
ERR409887       SAMEA2300506    ERX376258       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409887/ERR409887.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409887/C_21.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409887     
ERR409890       SAMEA2300489    ERX376241       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409890/ERR409890.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409890/H_01.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409890     
ERR409896       SAMEA2300508    ERX376260       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409896/ERR409896.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409896/C_32.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409896     
ERR409891       SAMEA2300494    ERX376246       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409891/ERR409891.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409891/C_14.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409891     
ERR409900       SAMEA2300499    ERX376251       PRJEB5212       9606    Homo sapiens    ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409900/ERR409900.fastq.gz        ftp.sra.ebi.ac.uk/vol1/run/ERR409/ERR409900/C_37.fastq.gz       ftp.sra.ebi.ac.uk/vol1/err/ERR409/ERR409900

  • STEP 5: Select FASTQ files (tick boxes next to the file names) and click on “Get download script”. This will download a bash script (e.g., )

image-20241025-044132.png

  • 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/ERR409/ERR409878/ERR409878.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409879/ERR409879.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409880/ERR409880.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409881/ERR409881.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409882/ERR409882.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409883/ERR409883.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409884/ERR409884.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409885/ERR409885.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409886/ERR409886.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409887/ERR409887.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409888/ERR409888.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409889/ERR409889.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409890/ERR409890.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409891/ERR409891.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409892/ERR409892.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409893/ERR409893.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409894/ERR409894.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409895/ERR409895.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409896/ERR409896.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409897/ERR409897.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409898/ERR409898.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409899/ERR409899.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409900/ERR409900.fastq.gz

Now using the TextEdit or NotePad app, we will add the following lines to the top of the script - copy and paste the following to the above script:

#!/bin/bash -l
#PBS -N ENA_data_download
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=24:00:00

#work on current directory (folder)
cd $PBS_O_WORKDIR

You should have this:

#!/bin/bash -l
#PBS -N ENA_data_download
#PBS -l select=1:ncpus=2:mem=4gb
#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/ERR409/ERR409878/ERR409878.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409879/ERR409879.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409880/ERR409880.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409881/ERR409881.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409882/ERR409882.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409883/ERR409883.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409884/ERR409884.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409885/ERR409885.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409886/ERR409886.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409887/ERR409887.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409888/ERR409888.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409889/ERR409889.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409890/ERR409890.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409891/ERR409891.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409892/ERR409892.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409893/ERR409893.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409894/ERR409894.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409895/ERR409895.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409896/ERR409896.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409897/ERR409897.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409898/ERR409898.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409899/ERR409899.fastq.gz
wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR409/ERR409900/ERR409900.fastq.gz

STEP 6: Save the file and now let’s transfer it to the HPC. See below:

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

Windows PC: open file finder and type the address below to connect to your home directory in the HPC, and then browse to the /workshop/2024-2/session6_smallRNAseq/data/mydata folder

\\hpc-fs\home\

Mac: open file finder and press “command” + “k” to open prompt, then type the below command, and then browse to the /workshop/2024-2/session6_smallRNAseq/data/mydata folder

smb://hpc-fs/home/

  • Drag and drop the script into the /workshop/2024-2/session6_smallRNAseq/data/mydata folder

STEP 7: We will ensure the copied file from our laptop / desktop does not have unwanted characters. Let’s move to the data folder:

cd $HOME/workshop/2024-2/session6_smallRNAseq/data/mydata

How to use the dos2unix tool? Type:

dos2unix --help

Now let’s run dos2unix conversion. Note the filename may vary, so adjust the filename as appropriate.

dos2unix -n ena-file-download-selected-files-20241013-1123.sh ena-file-download-selected-files-20241013-1123.pbs

  • Note: If you create a file using Microsoft Excel, it is likely that it will add ASCII characters, use dos2unix to remove such characters.

Now we are ready to submit to the HPC cluster the script to download FASTQ files:

qsub ena-file-download-selected-files-20241013-1123.pbs

Monitor progress of job:

qjobs

  • Note: Downloading the above datasets will take about ~50 minutes.

Find in the link below alternative approaches to download data from SRA, BaseSpace or use the nf-core/fetchngs pipeline:

Data Download

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