Parameters

The command-line interface is installed as excavate-ht. Run the following in Terminal to print descriptions of parameters (also described below). .. code-block:: bash

excavate-ht –help excavate-ht generate –help excavate-ht pair –help

EXCAVATE-HT Modes

EXCAVATE-HT has two modes: generate and pair.

excavate-ht generate

The generate mode creates a complete gRNA library using inputted variant data.

Required arguments

--vcf: Path to the VCF file. Comma-separated if more than one (e.g., cell_line.vcf.gz,1000genomes.vcf.gz). If using a phased cell-line VCF, put that first. Required.
--var_type: Variant type: cell-line or population. Can specify multiple, comma-separated (e.g., cell-line,population1,population2). Required.
--chrom_fa: Path to the chromosome FASTA file for your locus of interest (e.g., chr1sequence.fasta). Required.
--locus: Genomic locus in format chr#:start-end. Required.
-o, --output_dir: Output directory for results. Folder name if you are already in the excavate folder. Required.

Cas protein and PAM configuration

You must specify either --cas OR both --pam-list and --orient.

--cas: Specify Cas protein. One of: SpCas9, SpCas9_NG, enAsCas12a, or SaCas9.
--pam-list: PAM sequences (if not using a supported Cas protein). Comma-separated. Use IUPAC codes. Required if --cas is not specified.
--orient: PAM orientation. Choices: 3prime or 5prime. Required if --pam-list is specified.

Guide design parameters

--af-threshold: Allele frequency threshold between 0 and 1. A buffer of 0.01 is subtracted from the AF threshold to account for rounded values in VCF files. Default: 0.1
-g, --guide-length: Guide length in base pairs. Default: 20
-m, --max_snppos_in_protospacer: Maximum distance (bp) of SNP from PAM sequence. Default: 10

Off-target analysis

--off-targets: Enable off-target analysis: counts exact matches genome-wide and 1-bp mismatches in the chromosome of interest. Uses Bowtie1. Flag (no value needed).
--download-hg38-index: Download prebuilt hg38 Bowtie indexes (GRCh38_noalt_as) into <outdir>/bowtie_index and use them automatically. Flag (no value needed).
--genome-index-prefix: Bowtie1 index prefix for the genome FASTA (e.g., /path/to/index/hg38_bt1). If missing or index files are not found, EXCAVATE-HT will build it next to this prefix.
--genome_fa: Path to the whole genome FASTA file for your organism. Required if building Bowtie indexes from scratch (when not using --download-hg38-index or --genome-index-prefix).
--chrom-index-prefix: Bowtie1 index prefix for the chromosome FASTA (e.g., /path/to/index/chr1_bt1). If missing or index files are not found, EXCAVATE-HT will build it next to this prefix.
--bowtie-threads: Number of threads for Bowtie1. Defaults to NSLOTS if set (HPC environments), otherwise 4.

Output options

--split-phased: Enable splitting of gRNA libraries by cell-line phasing. Flag (no value needed).
--summary: Output a summary table for each gRNA library. Flag (no value needed).
--per-vcf: Save single-gRNA libraries for each VCF file, split by allele. Flag (no value needed).

Pairing options

--pairing-method

Enable pairing of gRNA to output a dual-guide library. Choices:

r: pair all guides that target different SNPs together (default).
fp: pair guides about a fixed point.
t: tiled pairing - pair guides that target adjacent variants.

Default: r

-f, --fixed-points-list

One or more fixed points, comma-separated (genomic coordinate without chr#, e.g., 11989251,12002042,...) in your locus to pair guides around. Required when using --pairing-method fp.

excavate-ht pair

The pair mode pairs an existing single-gRNA library to create a dual-guide library.