How to run RNAseq analysis in the computational infrastructure of INAB

Steps to run the analysis:

1. Log in to the appropriate server:

o 160.40.71.4

2. Identify where your NGS results (.fastq files) are located:

o e.g., /mnt/new_home/stamatopoulos_research/2025_05_21_ERIC_study

3. Move to the your working directory:

cd /work/folder_of_each_user

4. Create a new analysis directory:

mkdir RNAseq_analysis_2025_05_21

5. Move to the your new analysis directory:

cd /work/RNAseq_analysis_2025_05_21

6. Download script to your analysis directory

wget https://raw.githubusercontent.com/natanast/RNAseq_analysis/main/bash/star.sh

wget https://raw.githubusercontent.com/natanast/RNAseq_analysis/main/bash/runSTAR.sh 

7. Create a SampleList

You have to update with the right path to your fastq files.

ls -la /mnt/new_home/stamatopoulos_research/RNAseq_2025_05_21 | awk '{print $9}' | awk -F "_R" '{print $1}' | sort | uniq > SampleList

8. Remove samples that should not be analyzed:

nano SampleList

In order to edit the SampleList use the following:

o Delete button: Removes one letter

o Ctrl + k: Removes a whole row

o Ctrl + o and Enter: Saves the modified SampleList

o Ctrl + x: Exits and returns to the terminal

9. Run the analysis:

You have to update with the right path.

nohup bash runSTAR.sh /mnt/new_home/stamatopoulos_research/RNAseq_2025_05_21 SampleList /mnt/new_home/bio_tmp/HSapRefData/hg38/gencode/star /mnt/new_home/bio_tmp/HSapRefData/hg38/gencode/gencode.v47.primary_assembly.basic.annotation.gtf 16 &

10. After the analysis is completed

Move your folder from /work to /mnt/new_home

cd ..

rsync -r RNAseq_analysis_2025_05_21 /mnt/new_home/stamatopoulos_research