Visualization and quantification of two insertions within an amplicon.

[yxwvutt]

Hi there! I have a library that contains a very large amplicon with two different insertion sites, and I had two questions regarding using crispresso on this.

1. Is it possible to visualize the entire amplicon on the allele_frequency_table_around_sgRNA... files if it is very large? I would like to visualize at least where the two insertions begin and end with the constant region in between as well. Currently, I try to increase -plot_window_size to accomplish this, but I seem to hit an upper limit of how large I can make the window. The two insertions along with the constant the region in between is roughly 300 bp.

2. Is it possible to quantify two insertions at a time instead of one? One insertion is about ten times larger than the other. I believe my issue is related to what is being asked about in issue --quantification_window_coordinates (qwc): Bug - Cannot define multiple quant windows for multiple amplicons #371. Currently, I am only able to quantify the larger insertion, but I do at least see my smaller insertion on the same read sometimes manually.

[kclem]

Hi @yxwvutt,

1. CRISPResso is designed for visualizations of small insertions, and may not be appropriate for the visualization of deletions that are far apart. I'd recommend running with the default plot window size (20bp) but adding the parameters --bam_output (and optionally --bowtie2_index) and viewing the output bam in IGV.

2. What do you mean by quantifying two insertions at a time? If your deletions occur at sgRNA positions, a deletion in either position will make the read 'modified'. You can also use this handy script to quantify edits at different sgRNAs, and it reports whether reads were edited at one or both sgRNAs.

Feel free to reach out if this solution doesn't work.

Thanks,

Kendell