Tuxedo pipeline

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Step 1. fastq dump

  • This step is only required, when you want to using SRA fastq data
  fastq-dump -A SRR203363 SRR203363.sra 
  -A $sradata_accession $sra_data.sra  
  • if paired-end sequence file were used, we should use --split-3 option and check making *_1.fa and *_2.fa

Step 2. bowtie build (Build index file)

  /location_of_bowtie/bowtie-build reference.fa reference.fa

Step 3. tophat (calculate splice junction)

  /loaction_of_tophat/tophat -p number_of_threads \
  -G gff3_file_of_genome \
  -o tophat_ouput_dir \
  reference.fa \

Step 4. cufflinks - make gtf

  /location_of_cufflinks/cufflinks -p number_of_threads \
  -o cufflinks_out_dir \
  tophat_out_dir/accepted_hits.bam -g gfffile

Step 5. cuffmerge - merging gtf

  • locations of transcripts.gtf files derived by cufflinks should be listed in assembly.txt
  * in assembly.txt
  /location_of_cuffmerge/cuffmerge -g gff3_file_used_in_tophat \
  -s reference.fa \
  -p number_of_threads 

Step 6. cuffdiff - deg

  /cuffdiff_location/cuffdiff \
  -o cuffdiff_out_dir \
  -b reference.fa \
  -p number_of_threads \
  -L label_of_bam_1,label_of_bam_2 \
  -u gtf_file_derived_by_cuffmerge(merged_asm/transcript.gtf) \
  tophat_out_dir/accepted_hits.bam_1 tophat_out_dir/accepted_hits.bam_2

Step 7. cummeRbund - analysis

  • Execute R package in diff_out directory
  diff_out_dir/$ R 
  • Import cummeRbund package