After running SCRAP, use the indicated script command to perform peak calling. List the full paths to the directory containing the sample folders and the adapter file. Then, define the criteria for peak calling, including the minimum number of sequencing reads, and the number of distinct sequencing libraries needed for a peak to be recognized.
Indicate whether peaks must be contributed to by sncRNAs of the same family, which is relevant for miRNAs that share seed sequences and can bind to overlapping gene targets. Then, indicate the full path to the reference directory, MIR base abbreviation, and the reference genome abbreviation. After peak calling, run the peak annotation script.
Provide the full path to the resulting peaks. bed from the peak calling to the reference directory and the desired species for annotation. Use samtools merge to merge all the desired bam files to facilitate combined visualization.
Then, use the samtools sort for line-by-line sorting of merged. bam file. Index the sorted.
bam file using the samtools index, generating a binary samtools format index file required for genomic visualization tools. Finally, in the integrative genomics viewer, open the sorted. bam and indexed.
by file. The application of a modified version of SCRAP to previously published sequencing data sets revealed a decrease in miRNA interactions with Intron regions. The reduction was observed after isolating high confidence interactions through peak calling with SCRAP.
