trace-crispr 0.3.0

TRACE: Triple-aligner Read Analysis for CRISPR Editing

TRACE

Triple-aligner Read Analysis for CRISPR Editing

TRACE is a comprehensive tool for quantifying CRISPR editing outcomes from amplicon sequencing data. It combines multiple alignment strategies with k-mer classification to provide robust, accurate measurements of HDR, NHEJ, and other editing outcomes.

Features

• Triple-aligner consensus: Uses BWA-MEM, BBMap, and minimap2 for robust alignment
• Flexible input: Accepts DNA sequences directly or FASTA file paths
• Automatic inference: Detects PAM, cleavage site, homology arms, and edits from sequences
• Large edit support: Handles insertions up to 50+ bp with automatic k-mer size adjustment
• K-mer classification: Fast pre-alignment HDR/WT detection (auto-sizes k-mers based on edit)
• Multi-nuclease support: Cas9 and Cas12a (Cpf1) with correct cleavage geometry
• Auto-detection: Library type (TruSeq/Tn5), UMI presence, read merging need
• PCR deduplication: Automatic UMI-based (TruSeq) or position-based (Tn5) deduplication
• CRISPResso2 integration: Validation with standard CRISPR analysis tool

Installation

pip (Python package only)

pip install trace-crispr

conda (includes external aligners)

conda install -c bioconda -c conda-forge trace-crispr

Development installation

git clone https://github.com/k-roy/TRACE.git
cd TRACE
pip install -e ".[dev]"

Quick Start

TRACE accepts sequences as either DNA strings or FASTA file paths.

Example 1: Using FASTA files

trace run \
  --reference amplicon.fasta \
  --hdr-template hdr_template.fasta \
  --guide GCTGAAGCACTGCACGCCGT \
  --r1 sample_R1.fastq.gz \
  --r2 sample_R2.fastq.gz \
  --output results/

Example 2: Using DNA sequences directly

The reference amplicon will typically be longer than the HDR template so that the primers specifically amplify the target locus. This example shows a 250 bp reference sequence where the donor template is 150 bp long with the designed edit in the middle.

# Reference amplicon (250 bp) - includes flanking regions
# Guide sequence shown in lowercase for illustration
REF="ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG\
ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG\
gctgaagcactgcacgccgttgg\
ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG\
ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG"

# HDR template (150 bp) - centered on edit site
# Guide in lowercase, designed edit (T->A) shown in UPPERCASE
HDR="ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG\
gctgaagcactgcacgccgtAga\
ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG"

trace run \
  -r "$REF" \
  -h "$HDR" \
  -g GCTGAAGCACTGCACGCCGT \
  --r1 sample_R1.fastq.gz \
  --output results/

Note: The guide and edit are shown here in lowercase/uppercase for illustrative purposes. This formatting is not necessary - TRACE will automatically detect guide and edit positions from the sequences.

Check locus configuration without running

trace info \
  --reference amplicon.fasta \
  --hdr-template hdr_template.fasta \
  --guide GCTGAAGCACTGCACGCCGT

This will print:

============================================================
=== TRACE Analysis Configuration ===
============================================================

Reference sequence: 250 bp
HDR template: 150 bp
  - Template aligns at position 36 in reference

Donor template analysis:
  - Left homology arm: positions 36-110 on reference (75 bp)
  - Right homology arm: positions 113-185 on reference (73 bp)

  Edits detected (2 total):
    * Position 111: C -> G (substitution)
    * Position 112: C -> T (substitution)

Guide analysis:
  - Guide sequence: GCTGAAGCACTGCACGCCGT
  - Guide targets: positions 113-132 on reference (- strand)
  - PAM: CCC at positions 110-112 on reference
  - Cleavage site: position 116 on reference

Multiple samples

Create a sample key TSV:

sample_id	r1_path	r2_path	condition
sample_1	/path/to/S1_R1.fastq.gz	/path/to/S1_R2.fastq.gz	treatment
sample_2	/path/to/S2_R1.fastq.gz	/path/to/S2_R2.fastq.gz	control

Then run:

trace run \
  --reference amplicon.fasta \
  --hdr-template hdr_template.fasta \
  --guide GCTGAAGCACTGCACGCCGT \
  --sample-key samples.tsv \
  --output results/ \
  --threads 16

Per-sample locus sequences

For multiplexed experiments with different target loci, you can specify reference, hdr_template, and guide per-sample in the sample key TSV:

sample_id	r1_path	r2_path	condition	reference	hdr_template	guide
sample_1	/path/S1_R1.fq.gz	/path/S1_R2.fq.gz	treatment
sample_2	/path/S2_R1.fq.gz	/path/S2_R2.fq.gz	control
sample_3	/path/S3_R1.fq.gz	/path/S3_R2.fq.gz	treatment	locus2.fasta	locus2_hdr.fasta	ACGTACGTACGTACGTACGT
sample_4	/path/S4_R1.fq.gz	/path/S4_R2.fq.gz	control	locus2.fasta	locus2_hdr.fasta	ACGTACGTACGTACGTACGT

• Empty values: Use CLI defaults (--reference, --hdr-template, --guide)
• Filled values: Override CLI defaults for that sample
• Values can be DNA sequences or FASTA file paths

Using Cas12a

trace run \
  --reference amplicon.fasta \
  --hdr-template hdr_template.fasta \
  --guide GCTGAAGCACTGCACGCCGTAA \
  --nuclease cas12a \
  --sample-key samples.tsv \
  --output results/

Auto-Detection

TRACE automatically detects library characteristics to optimize analysis:

Library Type Detection

TRACE distinguishes between TruSeq (fixed-target amplicon) and Tn5 (tagmented locus) libraries by analyzing read alignment positions:

• TruSeq: Reads cluster at fixed start positions (primer binding sites)
• Tn5: Reads have scattered start positions (random Tn5 cutting)

Auto-detection results:
  - Library type: TruSeq (100% of reads cluster at fixed start position)
  - UMI detection: UMIs of length 6 bp detected
    --> Entering PCR deduplication mode...

UMI Detection

For TruSeq libraries, TRACE detects UMIs (Unique Molecular Identifiers) by analyzing sequence diversity at read starts:

• High diversity region = UMI
• Low diversity region = primer sequence
• Automatically determines UMI length (typically 4-12 bp)

Preprocessing Modes

Based on detection results, TRACE automatically selects the optimal preprocessing workflow:

Library	UMIs	Overlap (>=15bp)	Preprocessing	Output
TruSeq	Yes	Yes	dedup → trim → merge → collapse	merged FASTQ
TruSeq	Yes	No	dedup → trim	paired FASTQs
TruSeq	No	Yes	trim → merge → collapse	merged FASTQ
TruSeq	No	No	trim	paired FASTQs
Tn5	N/A	Yes	trim → merge → collapse → align → position dedup	merged FASTQ
Tn5	N/A	No	trim → align → position dedup	paired FASTQs

Example output:

Auto-detection results:
  - Library type: TruSeq (100% of reads cluster at fixed start position)
  - UMI detection: UMIs of length 6 bp detected
  - Read overlap: Enabled (~50bp overlap (25% of amplicon))
  - Preprocessing: dedup-trim-merge-collapse -> merged
    (UMI dedup -> trim -> merge -> collapse)
  - CRISPResso mode: merged (merged reads from preprocessing)

Designed Edit Detection

TRACE automatically detects the edits encoded in the donor by first aligning the HDR template to the reference. TRACE then classifies the intended edit as a single-nucleotide variant (SNV), multi-nucleotide variant (MNV), insertion, or deletion.

K-mers are selected that span the designed edit and are unique to the reference and donor. For large edits, TRACE automatically increases the k-mer size to ensure reliable classification. TRACE can handle MNVs or insertions with lengths up to the read length - 50 bp (e.g., 100 bp insertions can be detected with 150 bp reads).

Example output for a 20 bp insertion:

Edits detected (1 total):
  * Position 125: +ATCGATCGATCGATCGATCG (20 bp insertion)

Maximum edit size: 20 bp
Recommended k-mer size: 30 bp

Nuclease Support

Cas9 (SpCas9)

• PAM: NGG (3' of protospacer)
• Cleavage: 3 bp upstream of PAM (blunt ends)

Cas12a (LbCpf1)

• PAM: TTTN (5' of protospacer)
• Cleavage: 18-19 bp downstream on target strand, 23 bp on non-target
• Creates 4-5 nt 5' overhang (staggered cut)

Output

The main output is a TSV file with per-sample editing outcomes:

Column	Description
sample	Sample ID
classifiable_reads	Total classifiable reads
duplicate_rate	PCR duplicate rate
WT_%	Wild-type %
HDR_%	HDR %
NHEJ_%	NHEJ %
LgDel_%	Large deletion %
kmer_hdr_rate	K-mer method HDR rate
crispresso_hdr_rate	CRISPResso2 HDR rate
crispresso_indel_rate	CRISPResso2 indel rate

For Tn5/tagmented data or TruSeq amplicons with UMIs, TRACE will report on the PCR duplication rate and automatically perform deduplication:

• TruSeq with UMIs: Pre-alignment UMI-based deduplication
• Tn5: Post-alignment position-based deduplication

Dependencies

Python

• click>=8.0
• pysam>=0.20
• pandas>=1.5
• numpy>=1.20
• pyyaml>=6.0
• rapidfuzz>=3.0
• tqdm>=4.60

External tools (via conda)

• bwa>=0.7
• bbmap>=39
• minimap2>=2.24
• samtools>=1.16
• crispresso2 (optional, but enabled by default)

Author

Kevin R. Roy

License

MIT