# 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: 93.66%.
Guardrail Warning! Disproportionate percentages of reads were aligned to amplicon: HDR, Percent of aligned reads aligned to this amplicon: 6.31%.
Guardrail Warning! >=0.2% of substitutions were outside of the quantification window. Total substitutions: 3649, Substitutions outside window: 3500.

##### 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 21:37:32

Amplicon sequence:

    acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccatggtgcatctgactcctgTggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcaggttggtatcaaggtta

Guide sequence:

    TGCACCATGGTGTCTGTTTG

Command used:

    CRISPResso -r1 hdr.fastq.gz -a acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccatggtgcatctgactcctgTggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcaggttggtatcaaggtta -e acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccatggtgcaCctgactccGgaggagaagtctgccgttactgcGctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcaggttggtatcaaggtta -c atggtgcatctgactcctgTggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcag -g TGCACCATGGTGTCTGTTTG --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: acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccatggtgcatctgactcctgTggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcaggttggtatcaaggtta
    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: atggtgcatctgactcctgTggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcag
    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: acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccatggtgcaCctgactccGgaggagaagtctgccgttactgcGctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcaggttggtatcaaggtta
    fastp_command: fastp
    fastp_options_string:
    fastq_output: False
    fastq_r1: hdr.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: TGCACCATGGTGTCTGTTTG
    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:
    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. NHEJ reads align more closely to the unmodified reference sequence, but have mutations present in the specified quantification window. HDR reads align to the HDR reference sequence and have no mutations in the specified quantification window. Imperfect HDR reads have mutations in the specified window. AMBIGUOUS reads align equally well to the unmodified and HDR reference sequences.

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

##### Global frameshift analysis

Figure 5a: Frameshift analysis of coding sequence reads affected by modifications for all reads. Unmodified reference reads are excluded from this plot, and all HDR reads are included in this plot.

##### Global frameshift mutagenesis profiles

Figure 6a: Frameshift and in-frame mutagenesis profiles for all reads indicating position affected by modification. The y axis shows the number of reads and percentage of all reads in that category (frameshifted (top) or in-frame (bottom)). 2906 reads with no length modifications are not shown.

Data: Indel histogram for Reference

Data: Indel histogram for HDR

##### Global splicing analysis

Figure 8a: Predicted impact on splice sites for all reads. Potential splice sites modified refers to reads in which the either of the two intronic positions adjacent to exon junctions are disrupted.

##### HDR summary report (all reads aligned to Reference)

Figure 4g: 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 percentage table for all reads aligned to Reference

Data: Modification percentage table for all reads aligned to Reference

Data: Nucleotide frequency table for Reference

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

##### Amplicons