# CRISPResso2

### Analysis of genome editing outcomes from deep sequencing data

Guardrail Warning! Disproportionate percentages of reads were aligned to amplicon: Reference, Percent of aligned reads aligned to this amplicon: 57.59%.
Guardrail Warning! Disproportionate percentages of reads were aligned to amplicon: Prime-edited, Percent of aligned reads aligned to this amplicon: 38.94%.
Guardrail Warning! Disproportionate percentages of reads were aligned to amplicon: Scaffold-incorporated, Percent of aligned reads aligned to this amplicon: 2.12%.
Guardrail Warning! >=1.0% of reads have modifications at the start or end. Total reads: 24911, Irregular reads: 24907.
Guardrail Warning! >=0.2% of substitutions were outside of the quantification window. Total substitutions: 6550, Substitutions outside window: 5668.

##### CRISPResso2 run information

Figure 1a: The number of reads in input fastqs, after preprocessing, and after alignment to amplicons.

Data: Mapping statistics

CRISPResso version: 2.3.2

Run completed: 2025-04-25 21:37:06

Amplicon sequence:

    ACGTCTCATATGCCCCTTGGCAGTCATCTTAGTCATTACCTGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTAATGGTTGAGTTCCGTTTGTCTGCACAGCCTGAGACATTGCTGGAAATAAAGAAGAGAGAAAAACAATTTTAGTATTTGGAAGGGAAGTGCTATGGTCTGAATGTATGTGTCCCACCAAAATTCCTACGT

Command used:

    CRISPResso --fastq_r1 prime_editor.fastq.gz --amplicon_seq ACGTCTCATATGCCCCTTGGCAGTCATCTTAGTCATTACCTGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTAATGGTTGAGTTCCGTTTGTCTGCACAGCCTGAGACATTGCTGGAAATAAAGAAGAGAGAAAAACAATTTTAGTATTTGGAAGGGAAGTGCTATGGTCTGAATGTATGTGTCCCACCAAAATTCCTACGT --prime_editing_pegRNA_spacer_seq GTCATCTTAGTCATTACCTG --prime_editing_pegRNA_extension_seq AACGAACACCTCATGTAATGACTAAGATG --prime_editing_nicking_guide_seq CTCAACCATTAAGCAAAACAT --prime_editing_pegRNA_scaffold_seq GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC --write_cleaned_report --place_report_in_output_folder

Parameters:

    allele_plot_pcts_only_for_assigned_reference: False
    aln_seed_count: 5
    aln_seed_len: 10
    aln_seed_min: 2
    amplicon_min_alignment_score:
    amplicon_name: Reference
    amplicon_seq: ACGTCTCATATGCCCCTTGGCAGTCATCTTAGTCATTACCTGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTAATGGTTGAGTTCCGTTTGTCTGCACAGCCTGAGACATTGCTGGAAATAAAGAAGAGAGAAAAACAATTTTAGTATTTGGAAGGGAAGTGCTATGGTCTGAATGTATGTGTCCCACCAAAATTCCTACGT
    annotate_wildtype_allele:
    assign_ambiguous_alignments_to_first_reference: False
    auto: False
    bam_chr_loc:
    bam_input:
    bam_output: False
    base_editor_output: False
    bowtie2_index:
    coding_seq:
    config_file: None
    conversion_nuc_from: C
    conversion_nuc_to: T
    crispresso1_mode: False
    debug: False
    default_min_aln_score: 60
    disable_guardrails: False
    discard_guide_positions_overhanging_amplicon_edge: False
    discard_indel_reads: False
    dsODN:
    dump: False
    exclude_bp_from_left: 15
    exclude_bp_from_right: 15
    expand_allele_plots_by_quantification: False
    expand_ambiguous_alignments: False
    expected_hdr_amplicon_seq:
    fastp_command: fastp
    fastp_options_string:
    fastq_output: False
    fastq_r1: prime_editor.fastq.gz
    fastq_r2:
    file_prefix:
    flash_command: None
    flexiguide_gap_extend_penalty: -2
    flexiguide_gap_open_penalty: -20
    flexiguide_homology: 80
    flexiguide_name:
    flexiguide_seq: None
    force_merge_pairs: False
    guide_name:
    guide_seq:
    halt_on_plot_fail: False
    ignore_deletions: False
    ignore_insertions: False
    ignore_substitutions: False
    keep_intermediate: False
    max_paired_end_reads_overlap: None
    max_rows_alleles_around_cut_to_plot: 50
    min_average_read_quality: 0
    min_bp_quality_or_N: 0
    min_frequency_alleles_around_cut_to_plot: 0.2
    min_paired_end_reads_overlap: 10
    min_single_bp_quality: 0
    n_processes: 1
    name:
    needleman_wunsch_aln_matrix_loc: EDNAFULL
    needleman_wunsch_gap_extend: -2
    needleman_wunsch_gap_incentive: 1
    needleman_wunsch_gap_open: -20
    no_rerun: False
    output_folder:
    place_report_in_output_folder: True
    plot_histogram_outliers: False
    plot_window_size: 20
    prime_editing_gap_extend_penalty: 0
    prime_editing_gap_open_penalty: -50
    prime_editing_nicking_guide_seq: CTCAACCATTAAGCAAAACAT
    prime_editing_override_prime_edited_ref_seq:
    prime_editing_override_sequence_checks: False
    prime_editing_pegRNA_extension_quantification_window_size: 5
    prime_editing_pegRNA_extension_seq: AACGAACACCTCATGTAATGACTAAGATG
    prime_editing_pegRNA_scaffold_min_match_length: 1
    prime_editing_pegRNA_scaffold_seq: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC
    prime_editing_pegRNA_spacer_seq: GTCATCTTAGTCATTACCTG
    quantification_window_center: -3
    quantification_window_coordinates: None
    quantification_window_size: 1
    samtools_exclude_flags: 0
    save_also_png: False
    split_interleaved_input: False
    stringent_flash_merging: False
    suppress_amplicon_name_truncation: False
    suppress_plots: False
    suppress_report: False
    trim_sequences: False
    trimmomatic_command: None
    trimmomatic_options_string:
    use_legacy_insertion_quantification: False
    use_matplotlib: False
    verbosity: 3
    write_cleaned_report: True
    write_detailed_allele_table: False
    zip_output: False

Running log

##### Allele assignments

Figure 1b: Alignment and editing frequency of reads as determined by the percentage and number of sequence reads showing unmodified and modified alleles.

Data: Quantification of editing

Figure 1c: Alignment and editing frequency of reads as determined by the percentage and number of sequence reads showing unmodified and modified alleles.

Data: Quantification of editing

##### Prime editing report (all reads aligned to Reference)

Figure 11a: Nucleotide distribution across all amplicons. At each base in the reference amplicon, the percentage of each base as observed in sequencing reads is shown (A = green; C = orange; G = yellow; T = purple). Black bars show the percentage of reads for which that base was deleted. Brown bars between bases show the percentage of reads having an insertion at that position.

Data: Nucleotide frequency table for Reference

Data: Nucleotide frequency table for Prime-edited

Data: Nucleotide frequency table for Scaffold-incorporated

##### Prime editing summary plots at analysis positions (aligned to Reference)

Figure 11b: Nucleotide distribution around the PE Extension (AACGAACACCTCATGTAATGACTAAGATG).

Data: Nucleotide frequency in quantification window for Reference

Data: Nucleotide frequency in quantification window for Prime-edited

Data: Nucleotide frequency in quantification window for Scaffold-incorporated

Figure 11b: Nucleotide distribution around the PE spacer sgRNA (GTCATCTTAGTCATTACCTG).

Data: Nucleotide frequency in quantification window for Reference

Data: Nucleotide frequency in quantification window for Prime-edited

Data: Nucleotide frequency in quantification window for Scaffold-incorporated

Figure 11b: Nucleotide distribution around the PE nicking sgRNA (CTCAACCATTAAGCAAAACAT).

Data: Nucleotide frequency in quantification window for Reference

Data: Nucleotide frequency in quantification window for Prime-edited

Data: Nucleotide frequency in quantification window for Scaffold-incorporated

##### Scaffold insertions

Figure 11c: Scaffold insertion lengths and deletion lengths in reads that contain a scaffold insertion. 'Length matching scaffold' shows the number of basepairs immediately after the pegRNA extension sequence that exactly match the scaffold RNA sequence. 'Insertion length' shows the length of the insertion immediately after the pegRNA extension sequence (including bases that do not match the scaffold sequence).

Data: Scaffold insertion alleles with insertion sizes

Reads are aligned to each amplicon sequence separately. Quantification and visualization of these reads are shown for each amplicon below:

##### Amplicons