CRISPResso2 does not correspond to IGV results

[zhaozhaocuicui]

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[zhaozhaocuicui]

The first image is the result of CRISPResso2, and the second image is the result of IGV. The two results do not correspond. The IGV result shows the missing AAGGC, but the CRISPResso result shows AGGCA

[Colelyman]

Hi @zhaozhaocuicui,

Thanks for using CRISPResso and for asking this question! As you may know, the CRISPResso allele plot that you have included is just the most frequently occurring alleles, so the reads and order shown in IGV may not correspond fully. If you want to do a full comparison, I would recommend looking at the Alleles_frequency_table_around_sgRNA_*.txt file, which will have all of your reads (including the reads with low frequency).

Would you be able to provide what aligner you used prior to visualizing in IGV?

If you would like us to take a closer look at what is going on, you can send the input fastq and CRISPResso command that you used, either via this issue or [email protected] for more privacy.

Thanks, Cole

[zhaozhaocuicui]

Hello, thank you for your @Colelyman I only used the following two commands before: 1.minimap2 -O 4 -E 1 -Y -ax map-ont --secondary=no -t 5 /data03/zzw/DAP-seq/Gmax_508_Wm82_bowtie2DB/Gmax_508_v4.0.fa "$fastq_file" > "$output_sam" 2. CRISPRessoWGS
--needleman_wunsch_gap_open -5
--needleman_wunsch_gap_extend 0
-b "$bam_in"
-f "$bed"
-r "$ref"
--exclude_bp_from_right 0
--exclude_bp_from_left 0
--plot_window_size 50
--cleavage_offset "$offset"
--ignore_insertions
--ignore_substitutions
--name "$out_name"
--output_folder "$out_dir"

[kclem]

Hi @zhaozhaocuicui,

Thanks for using CRISPResso.

Just to clarify, CRISPResso and IGV display different types of information, which is why the visualizations don’t look identical:

• IGV shows individual raw reads, typically sorted by their left-most alignment position.
• CRISPResso, on the other hand, clusters reads into allele groups and displays them sorted by allele frequency, with the most common allele on top.

Looking at your example, the results are actually consistent between the two tools.

• In the IGV coverage track, the most prominent indel signature is a 5bp deletion (visible as the half-height gray bars), which matches the top allele in CRISPResso at 27.7%.
• The next most frequent signature is a 1bp deletion, which CRISPResso reports at ~25%, and you can see this reflected in IGV as the short gray bar near the top of the coverage plot.

In summary, IGV shows individual reads; CRISPResso shows summarized allele frequencies.

Let us know if that doesn't make sense or if you have any other questions.