TwInsPEctor

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 a wiltype-edited compound reference.

Reads are categorized into one of the following eight allele types complete with detailed visualizations:

• Perfect TPE - complete programmed edit without indels.
• TPE Indel - complete programmed edit with indels.
• Left Flap - at least N consecutive programmed bases starting from the left but not from the right.
• Right Flap - at least N consecutive programmed bases starting from the right but not from the left.
• Imperfect TPE - incomplete programmed edit (neither or both flaps).
• Imperfect WT - incomplete wildtype sequence and none of the programmed edit.
• WT Indel - complete wildtype sequence with indels and none of the programmed edit.
• WT - complete wildtype sequence without indels and none of the programmed edit.

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 wildtype reference amplicon sequence including spacers.
-t, --twin_seq	full twin-pe reference amplicon sequence with 5′ and 3′ ends identical to the wildtype amplicon.
-g, --peg_spacers	comma-separated pegRNA spacer sequences: <spacerA>,<spacerB>

---

Optional Arguments

Argument	Description	Default
-r2, --fastq_r2	path to fastq r2 file for paired-end data.	None
-o, --output_root	root output directory for TwInsPEctor results. If not provided, a folder is created in the working directory based on input fastq names.	auto
-ne, --num_changes_to_check	minimum number of programmed bases that must be edited for classification.	2
-rcm, --recoding_mode	enable recoding mode when edits consist only of base substitutions.	off
-dmas, --default_min_aln_score	minimum homology score for CRISPResso2 to align read to compound reference.	50
-pfr, --plot_full_reads	display full read sequences in allele tables.	off
-ied, --ignore_extraspacer_deletions	ignore deletions outside the edit window (beyond spacers).	off
-nat, --no_allele_tables	skip generation of allele tables to reduce runtime.	off
-mfa, --min_frequency_alleles	minimum percent read frequency required to report an allele.	0.0
-mnr, --max_n_rows	maximum number of allele rows displayed in tables.	50
-nrr, --no_rerun	do not rerun CRISPResso2 if the same parameters were already completed.	off
-kco, --keep_crispresso_outputs	preserve CRISPResso2 output folders after analysis.	off
-ts, --trim_string	string to trim reads using fastp with override options within CRISPResso2 before analysis.	None
-fp, --fastp_command	command to run fastp for read trimming within CRISPResso2 before analysis.	None

Usage

TwInsPEctor -r1 <FASTQ_R1> [-r2 <FASTQ_R2>] -w <WT_SEQUENCE> -t <TWINPE_SEQUENCE> -g <PEG_SPACER_A>,<PEG_SPACER_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.