Linux_commands_Transcriptome_analysis

#Evolution of drug resistance in cancer cells involves generation of numerous mutations in non-coding genome that reduces the chances of DNA breaks
#Authors:
#Santosh Kumar1†, Valid Gahramanov1†, Julia Yaglom1, Shivani Patel1†, Lukasz Kaczmarczyk1, Ivan Alexandrov2, Gabi Gerlitz1, Mali Salmon-Divon1, Michael Y. Sherman1*
#Affiliations 
#1.	Department of Molecular Biology, Ariel University, Israel-40700.
#2.	Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia-119071.


#********Codes to be used for Transcriptome analysis in linux server to process raw reads from sequencing
cd ../

#Commands for FASTQC and trim galore


#To make excutable forloop
nano filename for making it as loop and write command
control x to exit
choose yes to save it

# Make it excutable
 chmod 755 filename
755 runs all file types 
Should show file in green colour now

#To run the command

bash qc_trimm 

for fileNum in {1..12}
do
        file=/home/SequencingData/kumar/Resistance_Irinotecan/BC_${fileNum}.fastq.gz
        nohup trim_galore --fastqc $file > ${fileNum}.out & 
done

$ top -u username to see the progress

#After trimming and quality control we run alignment of our fastq files to reference sequence

for fileNum in {1..12}
do
        file=/home/kumar/trim_galore_and_fastqc/BC_${fileNum}_trimmed.fq.gz
        ref=/home/Genomes/Human/hg38/UCSC/HISAT2/hg38_tran/genome_tran
        nohup hisat2 -x $ref -U $file 1> $fileNum.sam 2> $fileNum.out &

done 

#After we sam file format from alignment we need to convert that file to Bam file format for counting step
samtools view -S -b /home/kumar/Resistance_Irinotecan/1.sam -o 1.bam
#Run all codes by changing the sample numbers to convert to BAM

#After alignment we use output file of alignment to count the number of the reads per sample

featureCounts -T 16 -a /home/Genomes/Human/hg38/ensembl/Homo_sapiens.GRCh38.97.gtf -o featurecounts.txt /home/kumar/Resistance_Irinotecan/alignment_bam/1.bam
/............../home/kumar/Resistance_Irinotecan/alignment_bam/12.bam

#Last code will give featureCount file that will be used further in R environment to calculate differential expression of genes using edgeR.

#********End of codes******