CALITAS

This repository is home to CALITAS, a CRISPR-Cas-aware ALigner for In silico off-TArget Search. CALITAS implements a customized gapped alignment of guide sequences to genomes and other reference sequences, returning consistent and non-redundant alignments.

Overview

CALITAS is a suite of bioinformatic tools for enumerating candidate off-target sites for CRISPR guide sequences and standardizing their alignment. It features tools for searching an entire genome for candidate off-target sites, as well as for aligning guides to specific sequences or locations in a genome.

Key features of CALITAS include:

Detection of all candidate off-target sites up to the requested number of mismatches and gaps
Elimination of redundant alignments resulting in a single best or canonical alignment per locus
Searches with multiple PAM sequences and/or PAM-less searching
Integration of known variants in VCF format into genome-wide off-target searches
Customizable scoring system for weighting mismatches vs. gaps and differences in the protospacer vs. PAM

CALITAS is not intended to predict active off-target sites, but rather to enumerate candidate off-target sites for further investigation.

Getting CALITAS

Releases

Binary releases of CALITAS are available from the releases page on GitHub. The downloadable JAR files contain CALITAS and all dependencies, and need only a Java RunTime version 8 or higher installed. Once downloaded CALITAS can be run as follows to produce usage information:

java -Xmx8g -jar calitas.jar

Bioconda

Relases of CALITAS are also available via Bioconda. To use these releases you will first need to install the conda package manager - Miniconda is recommended if you do not already use conda. CALITAS can then be installed with:

conda install -c bioconda calitas

The conda release comes with a small helper script and can be run simply as calitas.

Building from Source

Both release and development versions of the code can be built from source. Builds are performed with sbt. Once sbt is installed run:

sbt clean assembly

This will build a JAR equivalent to a release JAR at calitas/target/scala-2.12/calitas.jar

Publication

A CALITAS manuscript is currently in review. This section will be updated with a reference to the publication when it is available.

Usage

CALITAS has four available sub-commands:

AlignToReference performs glocal alignment of query sequence to a window on the reference
PairwiseAlignSequences performs pairwise alignment of sequences
PrepareVcf prepares a VCF for optimal use by SearchReference
SearchReference searches a FASTA file for alignments of a guide+PAM(s)

It should be noted that commands which use a genome or reference FASTA file require that the FASTA file have both an index and a sequence dictionary. These can be generated using samtools as follows:

samtools faidx ref.fa
samtools dict -a <assembly-name> -s <species> -o ref.dict ref.fa

The following is an example of invoking SearchReference to find candidate off target sequences in the HG38 genome for a single guide and PAM (note that location and sequence of the PAM is indicated by providing guide sequence in upper case and PAM sequence in lower case):

calitas SearchReference \
  -i CTTGCCCCACAGGGCAGTAAnrg \
  -I myguide \
  -r hg38.fa \
  -o myguide.hits.txt \
  --max-guide-diffs 5 \
  --max-pam-mismatches 1 \
  --max-gaps-between-guide-and-pam 3

The last three parameters are optional and replicate the defaults. Additional options are available; detailed usage including all available parameters can be obtained by running calitas SearchReference.

The following is an example of running AlignToReference to produce standarized alignments at locations where guide(s) are known to align. The invocation will produce the single best alignment per query sequence and target location:

calitas AlignToReference -i input.txt -r hg38.fa -o output.txt --window-size 60

With the following being an example of the tab-delimited input file for AlignToReference:

id	query	chrom	position
1	CTTGCCCCACAGGGCAGTAAnrg	chr1	13358
2	CTTGCCCCACAGGGCAGTAAnrg	chr1	510578
3	CTTGCCCCACAGGGCAGTAAnrg	chr1	844033

The output of both SearchReference and AlignToReference is a tab-delimited text file with one row per candidate off-target site including the following columns:

column name	description
`guide_id`	Name/ID of guide.
`unpadded_guide_sequence`	The sequence of the guide used, unpadded.
`genome_build`	The assembly name of the searched genome (e.g. `HG38`).
`chromosome`	Chromosome for target sequence alignment (eg: `chr3`).
`coordinate_start`	Start of the unpadded target sequence in the genome, 0-based open ended, excluding PAM.
`coordinate_end`	End of the unpadded target sequence in the genome, 0-based open ended, excluding PAM.
`strand`	Either `+` or `-`. The reported strand is the strand of the genome which matches the guide sequence. E.g. if strand is reported as `+` this means the guide resembles the sequence on the top strand of the genome, and will bind to the bottom strand of the genome.
`unpadded_target_sequence`	The unpadded target sequence (as DNA) as found in the genome, without gaps/bulges, excluding PAM. Reported sequence matches the reported `strand` (i.e. `-` strand hits will report the reverse complement of the genomic sequence).
`ten_bases_5_prime`	The 10 bases from the reference genome immediately 5' of the off-target location (`coordinate_start`/`coordinate_end`), respecting `strand`.
`ten_bases_3_prime`	The 10 bases from the reference genome immediately 3' of the off-target location (`coordinate_start`/`coordinate_end`), respecting `strand`.
`pam_used`	PAM used in the alignment (eg: `nrg`).
`variant_id`	When searching using a VCF, a semi-colon separated list of variant IDs (e.g. `rs1234;rs2345`) that have non-reference alleles present in the off-target alignment. May be empty when no variants are present.
`variant_description`	When searching using a VCF, a semi-colon separated list of variant descriptions in the format `id:pos:ref>alt:af` where pos is the position within the alignment and af is the allele frequency of the alternate allele.
`variant_vcf`	When searching using a VCF, a string composed of filname of the VCF followed by a colon (`:`) and then the MD5 of the VCF.
`allele_frequency`	When searching using a VCF, the minimum allele frequency of any variant included in the target alignment.
`score`	Alignment score (including PAM).
`guide_mm`	Mismatches in the guide region (excluding PAM).
`guide_gaps`	Gaps in the guide region (excluding PAM).
`guide_mm_plus_gaps`	Total gaps and mismatches in the guide region (excluding PAM).
`pam_mm`	Mismatches in the PAM region.
`total_mm_plus_gaps`	Total count of mismatches and gaps across the both guide and PAM regions.
`padded_guide`	Guide + PAM sequence with padding for mismatches and bulges/gaps.
`padded_alignment`	Visual representation of the guide-target alignment: `
`padded_target`	Target sequence, including PAM, with padding for mismatches and bulges/gaps.
`padded_extra_8_bases_5_prime`	an additional 8 bases on the 5' side of the padded_target.
`padded_extra_8_bases_3_prime`	an additional 8 bases on the 3' side of the padded_target.
`cigar`	Cigar representation of guide sequence alignment.
`unpadded_guide_sequence_length`	Length of guide sequence not including PAM, nor gaps/bulges.
`unpadded_target_sequence_length`	Length of target sequence not including PAM, nor gaps/bulges.
`aligner`	Aligner name, e.g. `CALITAS:SearchReference`.
`aligner_version`	Version number of the CALITAS software used to produce the alignments.
`aligner_search_pam`	Comma-separated list of pams used during the search.
`aligner_other_parameters`	A semicolon-separated list of parameters provided when running CALITAS.
`time_stamp`	A date and time stamp for when the alignment run was started (UTC) in this format: Wed Jan 6 16:58:29 UTC 2021.

Note on coordinates produced by CALITAS

All genome coordinates in CALITAS output files are 0-based open ended, the same systemm used in BED files. I.e. the first base of a chromosome or sequence is represented by 0, and when describing a region on chromosome we specify the first base included in the interval as the start and the base after the interval as the end. E.g. the first 10bp on a chromosome would be represented as 0-10.

tfenne/calitas