TwInsPEctor is a tool for analyzing twin prime editing outcomes from next-generation sequencing reads, including data processing and visualization.

It utilizes CRISPResso2 for alignment of reads to wild type, edited, and wildtype-edited composite references.

Reads are classified into one of ten allele outcome categories:

• Perfect TPE - exact programmed edit.
• Dual Flap - meets both Flap A and Flap B requirements.
• Flap A - at least N contiguous bases from the start of only the pegRNA-a templated sequence.
• Flap B - at least N contiguous bases from the start of only the pegRNA-b templated sequence.
• Flap A Hybrid - meets Flap A requirements but retains wild type sequence from the opposing 5' flap.
• Flap B Hybrid - meets Flap B requirements but retains wild type sequence from the opposing 5' flap.
• Imperfect TPE - contains a partially edited sequence meeting none of the flap category requirements.
• Null - lacks any detectable wild type or edited sequence in between the two pegRNA nick sites.
• Imperfect WT - partial wildtype sequence with none of the edited sequence.
• WT - unedited wild type sequence.

Features

• Command-line interface for streamlined analysis
• Data processing utilities for twin prime editing experiments
• Visualization tools for editing outcomes

Installation

Typical installation time on a normal desktop computer is less than 5 minutes.

Using Conda

conda install bioconda::twinspector

From Source

git clone https://github.com/clementlab/TwInsPEctor.git
cd TwInsPEctor
pip install .

Overview of command line interface

Required Arguments

Argument	Description
-r1, --fastq_r1	Path to FASTQ R1 file.
-w, --wt_seq	Full wild-type reference amplicon sequence including spacers.
-t, --tpe_seq	Full Twin prime edited reference amplicon sequence with 5' & 3' ends identical to wildtype reference amplicon.
-g, --peg_spacers	Comma-separated pegRNA spacer sequences: <spacer A>,<spacer B>. Should include bases immediately adjacent to but not including the PAM sequence (usually 20nt 5' of NGG).

Recommended Arguments

Argument	Description
-rt, --rt_templates	Comma-separated pegRNA reverse transcriptase templates: <RT template A>,<RT template B>. Informs flap analysis and plotting.

Optional Arguments

Argument	Description	Default
-r2, --fastq_r2	Path to FASTQ R2 file for paired-end data.	None
-o, --output_root	Root output folder for CRISPResso2 and TwInsPEctor results. If not provided, a folder will be created in the current working directory based on the input FASTQ file names.	auto
-rcm, --recoding_mode	Run in recoding mode if the wild-type and twin prime edited sequences are the same length and should be evaluated as having only base substitutions.	off
-ne, --min_num_base_edits	Minimum number of twinPE-matched base changes required for a read to be considered edited.	3 (replacement) 2 (recoding)
-coa, --cleavage_offset_a	Cleavage offset for pegRNA spacer A.	-3
-cob, --cleavage_offset_b	Cleavage offset for pegRNA spacer B.	-3
-dmas, --default_min_aln_score	Default alignment score against any reference for a read to be included in analysis.	30
-pfr, --plot_full_reads	Display full read sequences in allele tables.	off
-ncda, --no_collapse_displayed_alleles	Do not combine alleles that become identical in the displayed allele table window.	off
-naa, --no_alignment_adjustments	Do not adjust for homologies in the allele tables. This does not affect allele classification.	off
-ied, --ignore_extraspacer_deletions	Classification ignores deletions occurring beyond the spacers (outside edit window).	off
-nf, --no_figures	Skip all figures if only text outputs are desired.	off
-nsf, --no_summary_figures	Skip summary barplots and summarized allele tables.	off
-nbf, --no_per_base_figures	Skip per-base barplots.	off
-nmf, --no_mutation_figures	Skip mutation barplots.	off
-pet, --plot_extended_tables	Generates separate allele tables for each category. Controlled by --max_n_rows and --min_frequency_alleles.	off
-pdf, --save_pdf	Only save PDF versions of all plots.	off
-npng, --no_save_png	Do not save PNG versions of all plots.	off
-mfa, --min_frequency_alleles	Minimum percent read frequency required to report an allele in the allele tables.	0.1
-mnr, --max_n_rows	Maximum number of allele rows to display in the allele tables by category.	25
-mna, --max_n_alleles_to_write	Maximum number of alleles per category to write to the f7 text file.	50
-nrr, --no_rerun	Do not rerun CRISPResso2 if a run using the same parameters has already been finished.	off
-kco, --keep_crispresso_outputs	Do not delete CRISPResso2 output folders after analysis.	off
--crispresso_args	Additional arguments to pass to CRISPResso2 (wrapped in quotes); do not include --n_processes.	""
-p, --n_processes, --n_threads	Total process budget. CRISPResso2 divides it across reference runs; allele-table plotting uses up to N processes.	1
-v, --verbose	Print verbose CRISPResso2 output.	off

Usage

TwInsPEctor -r1 <FASTQ_R1> [-r2 <FASTQ_R2>] -w <WT_SEQUENCE> -t <TWINPE_SEQUENCE> -g <PEG_SPACER_A>,<PEG_SPACER_B> -rt RT_TEMPLATE_A>,<RT_TEMPLATE_B> [options]

After installation, use the CLI for help:

TwInsPEctor --help

Or run the main module directly:

python -m TwInsPEctor

Requirements

• Python >=3.8
• CRISPResso2 (installed via Bioconda)

System Requirements

• Operating Systems: Linux, macOS
• Tested on: Rocky Linux 8.10
• Hardware Requirements: This software can run on a standard desktop computer and does not require any non-standard hardware.

Demo

For instructions on running TwInsPEctor on the provided demo data, including expected outputs, please see demo/demo.md.

Expected run time for the demo on a normal desktop computer is < 1 minute.

License

This project is licensed under the MIT License. See the LICENSE file for details.

Authors

• Nate Masson
• Kendell Clement

Dependency Notice

This software requires CRISPResso2 to be installed separately.

CRISPResso2 is distributed under its own license terms, which may restrict commercial use.

Users are responsible for ensuring compliance with the CRISPResso2 license when using this software.

This project does not redistribute CRISPResso2 and does not grant any rights to it.