# CRISPResso2

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

##### nhej

Guardrail Warning! >=1.0% of reads have modifications at the start or end. Total reads: 24344, Irregular reads: 458.
Guardrail Warning! >=0.2% of substitutions were outside of the quantification window. Total substitutions: 9093, Substitutions outside window: 1051.

##### 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 21:37:44

Amplicon sequence:

    AATGTCCCCCAATGGGAAGTTCATCTGGCACTGCCCACAGGTGAGGAGGTCATGATCCCCTTCTGGAGCTCCCAACGGGCCGTGGTCTGGTTCATCATCTGTAAGAATGGCTTCAAGAGGCTCGGCTGTGGTT

Command used:

    CRISPResso -r1 nhej.r1.fastq.gz -r2 nhej.r2.fastq.gz -a AATGTCCCCCAATGGGAAGTTCATCTGGCACTGCCCACAGGTGAGGAGGTCATGATCCCCTTCTGGAGCTCCCAACGGGCCGTGGTCTGGTTCATCATCTGTAAGAATGGCTTCAAGAGGCTCGGCTGTGGTT -n nhej --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: AATGTCCCCCAATGGGAAGTTCATCTGGCACTGCCCACAGGTGAGGAGGTCATGATCCCCTTCTGGAGCTCCCAACGGGCCGTGGTCTGGTTCATCATCTGTAAGAATGGCTTCAAGAGGCTCGGCTGTGGTT
    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: nhej.r1.fastq.gz
    fastq_r2: nhej.r2.fastq.gz
    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: nhej
    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:
    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:
    prime_editing_pegRNA_scaffold_min_match_length: 1
    prime_editing_pegRNA_scaffold_seq:
    prime_editing_pegRNA_spacer_seq:
    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

##### Nucleotide composition

Hover your mouse over the bottom image to zoom in on a specific region.
Figure 2a: Nucleotide distribution across amplicon. 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

##### Modification lengths

Figure 3a: Frequency distribution of alleles with indels (blue) and without indels (red). Note that histograms are clipped to show 99% of the data. To show all data, run using the parameter '--plot_histogram_outliers'. (Maximum 85 not shown) (Minimum -53 not shown)

Data: Indel histogram

Figure 3b: Left panel, frequency distribution of sequence modifications that increase read length with respect to the reference amplicon, classified as insertions (positive indel size). Middle panel, frequency distribution of sequence modifications that reduce read length with respect to the reference amplicon, classified as deletions (negative indel size). Right panel, frequency distribution of sequence modifications that do not alter read length with respect to the reference amplicon, which are classified as substitutions (number of substituted positions shown).

Data: Insertions frequency

Data: Deletions Frequency

Data: Substitutions Frequency

##### Indel characterization

Figure 4a: Combined frequency of any modification across the amplicon. Modifications outside of the quantification window are also shown.
Figure 4b: Frequency of insertions, deletions, and substitutions across the entire amplicon, including modifications outside of the quantification window.

Data: Modification frequency

Figure 4c: Frequency of insertions, deletions, and substitutions across the entire amplicon, considering only modifications that overlap with the quantification window.

Data: Modification frequency in quantification window

Figure 4d: Position dependent insertion size(left) and deletion size (right), including only modifications that overlap with the quantification window.