Inquiry about Chimeric Junction Identification in scRNA-seq Data Analysis

[luojinqin]

Hello,
Thank you very much for the tool you developed; it has helped me a lot.I am writing to seek clarification regarding the identification of chimeric junctions in the context of analyzing circRNAs in scRNA-seq data.

In my current work, I am aiming to identify circRNAs in different cell subpopulations using the following workflow:

1、Splitting the total BAM file into smaller BAM files based on cell type barcodes, resulting in, for example, two smaller BAM files, each
2、representing a distinct cell type.
3、Converting the smaller BAM files back into FASTQ format.
4、Utilizing STAR to separately align the small FASTQ files and obtain chimeric out junctions.
5、Employing the chimeric out junctions in conjunction with the CircExplorer tool to identify circRNAs.

During the execution of this workflow, I have encountered an issue where the chimeric out junction file obtained from reconstructing FASTQ files from the total BAM differs from the combined result of identifying chimeric junctions after alignment of the split BAM files. This inconsistency has prompted me to inquire: Does the identification of chimeric junctions potentially vary based on the overall library composition? Could it be that the determination of whether a read represents a chimeric junction is not independent for each read?

For example, after running STAR on BAM files from two clusters, 844,772 and 214,489 chimeric junction reads were identified, respectively. When these BAM files were merged, a total of 1,820,801 chimeric junction reads were identified(fig.1). However, when feeding these three chimeric junction files into CircExplorer2 for circRNA identification, the number of circRNAs detected in each of the two clusters individually was higher than the result obtained after merging the files(fig.2).

I am interested in understanding the potential factors influencing chimeric junction identification and would greatly appreciate any insights you could provide on this matter.

Thank you for your time and attention. I eagerly await your response.