Difference between revisions of "Tuxedo pipeline"
From Crop Genomics Lab.
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=== Step 1. fastq dump === | === Step 1. fastq dump === | ||
| − | fastq-dump -A SRR203363 SRR203363.sra | + | * This step is only required, when you want to using SRA fastq data |
| − | -A $sradata_accession $sra_data.sra | + | fastq-dump -A SRR203363 SRR203363.sra |
| − | if paired-end sequence file were used, we should use --split-3 option and check making *_1.fa and *_2.fa | + | -A $sradata_accession $sra_data.sra |
| − | + | ||
| − | === Step 2. bowtie build ( | + | * if paired-end sequence file were used, we should use --split-3 option and check making *_1.fa and *_2.fa |
| − | bowtie-build | + | |
| − | + | === Step 2. bowtie build (Build index file) === | |
| − | === Step 3. tophat | + | /location_of_bowtie/bowtie-build reference.fa reference.fa |
| − | / | + | |
| − | + | === Step 3. tophat (calculate splice junction) === | |
| + | /loaction_of_tophat/tophat -p number_of_threads \ | ||
| + | -G gff3_file_of_genome \ | ||
| + | -o tophat_ouput_dir \ | ||
| + | reference.fa \ | ||
| + | fastq_file | ||
| + | |||
=== Step 4. cufflinks - make gtf === | === Step 4. cufflinks - make gtf === | ||
| − | cufflinks -p | + | /location_of_cufflinks/cufflinks -p number_of_threads \ |
| − | + | -o cufflinks_out_dir \ | |
| + | tophat_out_dir/accepted_hits.bam -g gfffile | ||
| + | |||
=== Step 5. cuffmerge - merging gtf === | === Step 5. cuffmerge - merging gtf === | ||
| − | in | + | * locations of transcripts.gtf files derived by cufflinks should be listed in assembly.txt |
| + | |||
| + | * in assembly.txt | ||
| + | cufflinks_out_dir/transcript.gtf | ||
| + | cufflinks_out_dir/transcript.gtf | ||
| + | |||
| + | /location_of_cuffmerge/cuffmerge -g gff3_file_used_in_tophat \ | ||
| + | -s reference.fa \ | ||
| + | -p number_of_threads | ||
| + | assemblies.txt | ||
| − | |||
| − | |||
=== Step 6. cuffdiff - deg === | === Step 6. cuffdiff - deg === | ||
| − | / | + | /cuffdiff_location/cuffdiff \ |
| − | + | -o cuffdiff_out_dir \ | |
| + | -b reference.fa \ | ||
| + | -p number_of_threads \ | ||
| + | -L label_of_bam_1,label_of_bam_2 \ | ||
| + | -u gtf_file_derived_by_cuffmerge(merged_asm/transcript.gtf) \ | ||
| + | tophat_out_dir/accepted_hits.bam_1 tophat_out_dir/accepted_hits.bam_2 | ||
| + | |||
=== Step 7. cummeRbund - analysis === | === Step 7. cummeRbund - analysis === | ||
| − | R | + | * Execute R package in diff_out directory |
| − | + | diff_out_dir/$ R | |
| − | + | * Import cummeRbund package | |
| + | library(cummeRbund) | ||
| + | * [[cummeRbund command]] | ||
Latest revision as of 00:30, 2 January 2018
Contents |
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 $sra_data.sra
- if paired-end sequence file were used, we should use --split-3 option and check making *_1.fa and *_2.fa
Step 2. bowtie build (Build index file)
/location_of_bowtie/bowtie-build reference.fa reference.fa
Step 3. tophat (calculate splice junction)
/loaction_of_tophat/tophat -p number_of_threads \ -G gff3_file_of_genome \ -o tophat_ouput_dir \ reference.fa \ fastq_file
Step 4. cufflinks - make gtf
/location_of_cufflinks/cufflinks -p number_of_threads \ -o cufflinks_out_dir \ tophat_out_dir/accepted_hits.bam -g gfffile
Step 5. cuffmerge - merging gtf
- locations of transcripts.gtf files derived by cufflinks should be listed in assembly.txt
* in assembly.txt cufflinks_out_dir/transcript.gtf cufflinks_out_dir/transcript.gtf
/location_of_cuffmerge/cuffmerge -g gff3_file_used_in_tophat \ -s reference.fa \ -p number_of_threads assemblies.txt
Step 6. cuffdiff - deg
/cuffdiff_location/cuffdiff \ -o cuffdiff_out_dir \ -b reference.fa \ -p number_of_threads \ -L label_of_bam_1,label_of_bam_2 \ -u gtf_file_derived_by_cuffmerge(merged_asm/transcript.gtf) \ tophat_out_dir/accepted_hits.bam_1 tophat_out_dir/accepted_hits.bam_2
Step 7. cummeRbund - analysis
- Execute R package in diff_out directory
diff_out_dir/$ R
- Import cummeRbund package
library(cummeRbund)
