Tuxedo pipeline

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 (align preprocessing)

bowtie-build Gmax_189.fa Gmax_189.fa

Step 3. tophat - calculate splice junction

/data/program/tophat-1.4.1.Linux_x86_64/tophat -p 4(core) -G /data/ref/Gmax_189_gene.gff3 -o SRR203363_thout Gmax_189.fa(bowtie_index) SRR203363.fastq(reads)

Step 4. cufflinks - make gtf

cufflinks -p 4 -o ./SRR203363_clout(cufflinks_out_dir) SRR203363_thout/accepted_hits.bam(tophat_output)

Step 5. cuffmerge - merging gtf

• locations of transcripts.gtf files derived by cufflinks should be listed in assembly.txt

  /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)