CRISPRessoPooled
CRISPRessoPooled is a utility to analyze and quantify targeted sequencing CRISPR/Cas9 experiments involving pooled amplicon sequencing libraries. One common experimental strategy is to pool multiple amplicons (e.g. a single on-target site plus a set of potential off-target sites) into a single deep sequencing reaction1. CRISPRessoPooled demultiplexes reads from multiple amplicons and runs the CRISPResso utility with appropriate reads for each amplicon separately.
Modes
This tool can run in 3 different modes:
CRISPRessoPooled Parameters
CRISPRessoPooled Examples
CRISPRessoPooled‑‑fastq_r1 ‑‑fastq_r2 ‑‑amplicon_seq ‑‑amplicon_name ‑‑amplicon_min_alignment_score ‑‑default_min_aln_score ‑‑expand_ambiguous_alignments ‑‑assign_ambiguous_alignments_to_first_reference ‑‑guide_seq ‑‑guide_name ‑‑flexiguide_seq ‑‑flexiguide_homology ‑‑flexiguide_name ‑‑discard_guide_positions_overhanging_amplicon_edge ‑‑expected_hdr_amplicon_seq ‑‑coding_seq ‑‑config_file ‑‑min_average_read_quality ‑‑min_single_bp_quality ‑‑min_bp_quality_or_N ‑‑file_prefix ‑‑name ‑‑suppress_amplicon_name_truncation ‑‑output_folder ‑‑verbosity ‑‑split_interleaved_input ‑‑trim_sequences ‑‑trimmomatic_command ‑‑trimmomatic_options_string ‑‑flash_command ‑‑fastp_command ‑‑fastp_options_string ‑‑min_paired_end_reads_overlap ‑‑max_paired_end_reads_overlap ‑‑stringent_flash_merging ‑‑quantification_window_size ‑‑quantification_window_center ‑‑exclude_bp_from_left ‑‑exclude_bp_from_right ‑‑use_legacy_insertion_quantification ‑‑ignore_substitutions ‑‑ignore_insertions ‑‑ignore_deletions ‑‑discard_indel_reads ‑‑needleman_wunsch_gap_open ‑‑needleman_wunsch_gap_extend ‑‑needleman_wunsch_gap_incentive ‑‑needleman_wunsch_aln_matrix_loc ‑‑plot_histogram_outliers ‑‑plot_window_size ‑‑min_frequency_alleles_around_cut_to_plot ‑‑expand_allele_plots_by_quantification ‑‑allele_plot_pcts_only_for_assigned_reference ‑‑quantification_window_coordinates ‑‑annotate_wildtype_allele ‑‑keep_intermediate ‑‑dump ‑‑write_detailed_allele_table ‑‑fastq_output ‑‑bam_output ‑‑bowtie2_index ‑‑zip_output ‑‑max_rows_alleles_around_cut_to_plot ‑‑suppress_report ‑‑place_report_in_output_folder ‑‑suppress_plots ‑‑base_editor_output ‑‑conversion_nuc_from ‑‑conversion_nuc_to ‑‑prime_editing_pegRNA_spacer_seq ‑‑prime_editing_pegRNA_extension_seq ‑‑prime_editing_pegRNA_extension_quantification_window_size ‑‑prime_editing_pegRNA_scaffold_seq ‑‑prime_editing_pegRNA_scaffold_min_match_length ‑‑prime_editing_nicking_guide_seq ‑‑prime_editing_override_prime_edited_ref_seq ‑‑prime_editing_override_sequence_checks ‑‑crispresso1_mode ‑‑dsODN ‑‑auto ‑‑debug ‑‑no_rerun ‑‑n_processes ‑‑bam_input ‑‑bam_chr_loc ‑‑skip_failed ‑‑crispresso_command ‑‑amplicons_file ‑‑gene_annotations ‑‑bowtie2_options_string ‑‑use_legacy_bowtie2_options_string ‑‑min_reads_to_use_region_pooled ‑‑skip_reporting_problematic_regions ‑‑compile_postrun_references ‑‑compile_postrun_reference_allele_cutoff ‑‑alternate_alleles ‑‑limit_open_files_for_demux ‑‑aligned_pooled_bam ‑‑demultiplex_only_at_amplicons ‑‑demultiplex_genome_wide ‑‑disable_guardrails ‑‑use_matplotlib
Briefly, genomic DNA samples for pooled applications can be prepared by first amplifying the target regions for each gene/target of interest with regions of 150-400bp depending on the desired coverage. In a second round of PCR, with minimized cycle numbers, barcode and adaptors are added. With optimization, these two rounds of PCR can be merged into a single reaction. These reactions are then quantified, normalized, pooled, and undergo quality control before being sequenced.