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Transcriptome Analysis

1. install tophat
conda install tophat
2. install cufflinks
conda install cufflinks
3. install bowtie
conda install bowtie
4. Go to the folder M1 from the root and type the commands
5. indexing the genome
bowtie-build genome.fa
6. Align read to reference (step by step)
(here the input files are genes.gtf, genome.fa ,SRR671946.fastq, SRR671947.fastq,
SRR671948.fastq,SRR671948.fastq)(here reference genome annotation file : genes.gtf,
reference genome in fasta : genome.fa; SRR671946.fastq, SRR671947.fastq,
SRR671948.fastq,SRR671948.fastq => paired end sequence in 2 different condition
KNO3 and KCL)
(Output files: accepted_hits.bam=> mapped to reference genome file in .bam format;
unmapped.bam => not aligned with reference genome so further denovo assebly is
required)
tophat --no-coverage-search -p 2 -G genes.gtf -o SRR671946_tophatOut
genome.fa SRR671946.fastq
tophat --no-coverage-search -p 3 -G genes.gtf -o SRR671947_tophatOut
genome.fa SRR671947.fastq
tophat --no-coverage-search -p 3 -G genes.gtf -o SRR671948_tophatOut
genome.fa SRR671948.fastq
tophat --no-coverage-search -p 3 -G genes.gtf -o SRR671949_tophatOut
genome.fa SRR671949.fastq
7. Assemble Transcripts
cufflinks -p 3-o SRR671946_cufflinksout SRR671946_tophatOut/accepted_hits.bam
cufflinks -p 3-o SRR671947_cufflinksout SRR671947_tophatOut/accepted_hits.bam
cufflinks -p 3-o SRR671948_cufflinksout SRR671948_tophatOut/accepted_hits.bam
cufflinks -p 3-o SRR671949_cufflinksout SRR671949_tophatOut/accepted_hits.bam

8.Merge the transcripts to a comprehensive Transcriptome


(i) Create assembled_tc.txt file (contains file paths of the transcript
assemblies)
Example:
./SRR671946_cufflinksout/transcripts.gtf
./SRR671947_cufflinksout/transcripts.gtf
./SRR671948_cufflinksout/transcripts.gtf
./SRR671949_cufflinksout/transcripts.gtf
(ii) cuffmerge -g genes.gtf -s genome.fa -p 3 assembled_tc.txt (2 files :
genome.fa.fai; merged_asm )
9. enter in to merged_asm directory
cd merged_asm
ls (check for merged.gtf file in it)
10. Converting .gtf file to .fasta format
gffread merged.gtf -g /home/kiosk/Desktop/WGS/M1/genome.fa -w trans.fasta
11. To count no of sequences in trans.fasta files
grep -c "^>" trans.fasta
12. come to the previous folder M1
cd ..
13. Differential Expression under Different conditions (2 conditions kcl and KNO3)
cuffdiff -o diff_result -b gen ome.fa -p 2 -u -L
Root_Kcl_control,Root_KNO3_treatment merged_asm/merged.gtf
./SRR671946_tophatOut/accepted_hits.bam,./SRR671947_tophatOut/accepted_hits.bam
./SRR671948_tophatOut/accepted_hits.bam,./SRR671949_tophatOut/accepted_hits.bam
(Output files: diff_result folder => 23 files ; genes.fpkm_tracking (is an
important file to interppret))