junction.bed output files from tophat

Question

[blooming.daisy333]

im newbie to NGS and linux. I have used Tophat to determine the splice junction in my sequences. i want to know that how could i play with junctions.bed output file of tophat?

Actually I want to develop a PWM (position weight matrix) from this data. for which i need to extract 13 nucleotides (3 from exon and 10 from intron ) at 5' of splice junction and 17 nucleotides (14 intronic and 3 exonic nucleotides ) from 3' splice junction to buld PWM. can anyone help me how to extract these specific nucleotides in separate file? further is there any tool to deal with such questions???

[WouterDeCoster]

You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.

Please stop using Tophat https://t.co/Es4ohxOEyx Cole and I developed the method in *2008*. It was greatly improved in TopHat2 then HISAT & HISAT2. There is no reason to use it anymore. I have been saying this for years yet it has more citations this year than last #methodsmatter

— Lior Pachter (@lpachter) December 2, 2017

In addition, I converted this post from a "Tool" to a "Question". "Tool" should only be used to announce new software.

[blooming.daisy333]

thank you so much for the help...