How to generate all possible exonic BSJ events within transcripts taking 20 bp from each side of all exons and pasting them together in reverse order?
I would like to construct a library of Back splicing junctions (BSJ) from all the possible exons (reference file) from every human transcript for circRNA detection. For that, I need to extract 20-30 bp from each side and paste them together side by side in reverse order.
There are several reports which have done this.
I am attaching a paper that has used a similar approach.
Article Transcriptome-wide profiles of circular RNA and RNA-binding ...
I will be very grateful if someone can suggest ways to address this question.
Dear Vipin Bhardwaj
Your experimental plan seems logical but to speed up annotation of BSJ sites this tool might be useful for you-
https://circexplorer2.readthedocs.io/en/latest/tutorial/pipeline/
Good luck !
Dear Shambhabi Chatterjee Thank you for your suggestion. An apology that I did not mention it in my question, but I have tried various circRNA detection tools including circexplorer2 too. However, I would like to extend my approach to make all possible combinations of exons and would like to compare it to the existing circRNA detection tools. Thanks again for your time and response.
Dear Vipin Bhardwaj
As far as I know there is no way to do this for all possible combinations because we actually do not have a list of all circRNAs possible. Non coding RNA databases are constantly changing as new candidates and transcripts get added. Many of the transcripts also exhibit cell type and tissue specific expression. Therefore, as per my understanding you can properly design BSJ from the list of available and annotated circRNAs in CircBase / Circexplorer.
You might find some additional useful information in this paper, as they also constructed a BSJ -gRNA library - doi.org/10.1038/s41592-020-01011-4 . The supplemental files have detailed info.
The other option could be as you mentioned to do this manually one by one for all the possible exons (reference file). But this might also give you many false positives. So you would need carefully validate all BSJ and establish the respective circRNA transcripts later on.
Hope this was helpful.
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