RNA-Seq analysis

Biomix

• ssh -XC usernmae@biomix.dbi.udel.edu to log in
• scp /local/location/file.name.ext username@biomix.dbi.udel.edu:/target/folder to copy file to Biomix
• scp -r username@biomix.dbi.udel.edu:/target/file.name.ext /local/location to copy file from Biomix to local folder

Quality check

• srun -c 9 --mem=8000 --x11 /usr/local/FastQC/fastqc --threads 9 to check RNA-Seq quality using FastQC graphically
  • -c for CPU, --x11 for graphical window, --threads for CPU threads

Trimming

• sbatch trimming.slurm to trim off the adapter sequences using trim_galore
• squeue to check job status
• less slurm-123456.out to check for any errors or warnings
• less brain_a.s.fastq.gz_trimming_report.txt to check trimming report

Mapping

• sbatch mapping.slurm to map RNA-Seq data to human genome draft GRCh38 using hisat2
• samtools view brain_a.s_trimmed.bam | less to see the .bam file

Indexing

• sbatch bamindex.slurm uses samtools to index .bam files

Counting

• sbatch counting.slurm uses htseq-count for countring

Visualization and Analysis with R

• Import, filter, log transform, distance heatmap, PCA, gene heatmap, BCV plot, smear plot, differential expression analysis
• Codes are in the rscript