Difference between revisions of "Tuxedo pipeline"

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(Step 5. cuffmerge - merging gtf)
(Step 5. cuffmerge - merging gtf)
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* locations of transcripts.gtf files derived by cufflinks should be listed in assembly.txt
 
* locations of transcripts.gtf files derived by cufflinks should be listed in assembly.txt
  
   in assembly.txt
+
   * in assembly.txt
 
   cufflinks_out_dir/transcript.gtf
 
   cufflinks_out_dir/transcript.gtf
 
   cufflinks_out_dir/transcript.gtf
 
   cufflinks_out_dir/transcript.gtf

Revision as of 08:10, 11 April 2014

Contents

Step 1. fastq dump

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 \
  fastq_file

Step 4. cufflinks - make gtf

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

Step 5. cuffmerge - merging gtf

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

Step 6. cuffdiff - deg

  /cuffdiff_location/cuffdiff \
  -o out_dir \
  -b reference.fa \
  -p number_of_threads \
  -L label_of_bam_1,label_of_bam_2 \
  -u gtf_file_derived_by_cuffmerge \
  bam_1 bam_2

Step 7. cummeRbund - analysis

  • Execute R package in diff_out directory
  /diff_out/$ R 
  • Import cummeRbund package
  library(cummeRbund)