Tuxedo pipeline

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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

in the assemblies.txt, file location such as ./SRR####_clout/transcripts.gtf should be written in each line

/data/program/cufflinks-2.1.1.Linux_x86_64/cuffmerge -g /data/ref/Gmax_189_gene.gff3(used_at_tophat) -s ./Gmax_189.fa(bowtie_indexed_genome_fasta) -p 4 ./assemblies.txt

Step 6. cuffdiff - deg

/data/program/cufflinks-2.1.1.Linux_x86_64/cuffdiff -o ./diff_out/ -b Gmax_189.fa -p 4 -L w82Leaf,w82leaf -u ./merged_asm/merged.gtf ./SRR203363_thout/accepted_hits.bam ./SRR203363_thout/accepted_hits.bam

Step 7. cummeRbund - analysis

R Library(cummeRbund) Diff->