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Transcriptional Analysis Commands

The document outlines the steps to perform transcriptome analysis using Tophat, Cufflinks, and Cuffdiff. It involves aligning RNA-seq reads to a reference genome using Tophat, assembling transcripts using Cufflinks, merging the assemblies using Cuffmerge, and performing differential expression analysis using Cuffdiff to compare two conditions. The key steps are: 1) aligning reads from 4 samples to the reference genome and annotation file using Tophat, 2) assembling transcripts for each sample using Cufflinks, 3) merging the assemblies using Cuffmerge, 4) running Cuffdiff to analyze differential expression between two conditions.

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Sasikala Rajendran
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91 views2 pages

Transcriptional Analysis Commands

The document outlines the steps to perform transcriptome analysis using Tophat, Cufflinks, and Cuffdiff. It involves aligning RNA-seq reads to a reference genome using Tophat, assembling transcripts using Cufflinks, merging the assemblies using Cuffmerge, and performing differential expression analysis using Cuffdiff to compare two conditions. The key steps are: 1) aligning reads from 4 samples to the reference genome and annotation file using Tophat, 2) assembling transcripts for each sample using Cufflinks, 3) merging the assemblies using Cuffmerge, 4) running Cuffdiff to analyze differential expression between two conditions.

Uploaded by

Sasikala Rajendran
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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))

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