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steps.txt
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steps.txt
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1) Trimming
### USERS must provide their adapters or select from the lists of commonly used Illumina smRNA-seq adapters:
/mnt/niagads/users/yyee/data/illumina_smrna_truseq_3adapter.fas
/mnt/niagads/users/yyee/data/illumina_smrna_1.0_3adapter.fas
/mnt/niagads/users/yyee/data/illumina_smrna_1.5_3adapter.fas
/mnt/niagads/users/yyee/data/illumina_standard.fas
# need smrna_step_trim3.sh
# need run_cutadapt.sh
2) ## run STAR on the trimmed fastq files
INPUT FASTQ:
FASTQ files should be named as:
<tissue>_<SRR>_trimmed.fastq
<tissue>_<SRR>_rawreads.fastq
where <tissue> is, e.g., brain1, liver1, etc.
<SRR> is e.g. SRA run name SRR772426
bash run_star_smrna.sh fastq_samples_list_renamed
3) ## the above steps are for individual samples, if you want to perform per tissue study,
you can group the samples together as:
samtools merge adipose_star_hc.bam
star_m0_map100_adipose_SRR772426/Aligned.out.filtered.hardClipped.sorted.bam
star_m0_map100_adipose_SRR772427/Aligned.out.filtered.hardClipped.sorted.bam
# make sure you sort them for further analysis
samtools sort adipose_star_hc.bam adipose_star_hc_sorted
samtools index adipose_star_hc_sorted.bam
rm adipose_star_hc.bam
# create a list of bam files per individual samples
bam_list_star_hardclipped_samples
# create a list of bam files per tissues
bam_list_star_hardclipped_tissues
4) ## convert tissues' multi-mapped bam to bedgraphs
bash run_qsub.sh bam_to_bedgraph.sh bam_list_star_hardclipped_tissues
5) ## convert tissues' multi-mapped bedgraphs to bigwig
bash convert_bedgraph_to_bigwig.sh bam_list_star_multi_hardclipped_tissues
6) ## segmentation
bash segment_bedgraph.sh 'tissue'_star_hc_sorted.bam.pos.bedgraph > tissue_multi_pksem_pos.bed
bash segment_bedgraph.sh 'tissue'_star_hc_sorted.bam..neg.bedgraph > tissue_multi_pksem_neg.bed
7) ## annotation