Phased Assembly Variant Caller

Variant caller for assembled genomes.

Please discuss with authors prior to publishing results using PAV 3; contact contact information. A paper describing PAV 3 is in preparation.

Install and run

Install

PAV requires Python 3.12 or newer.

pip install pav3

Run

To run PAV, use the pav3 batch command after setting up configuration files (see below). A future version may add a command for single-assemblies without requiring configuration files.

Some Python environments may require you to run pav3 through the python command:

python -m pav3 batch

Containers

Public container images include PAV and all dependencies.

[!IMPORTANT] Pulling containers will generate 401 errors pending permissions changes by our organization. This should be corrected within a few days, please check back.

Docker:

docker pull ghcr.io/becklaboratory/pav3:latest

docker run --rm -v "$PWD:$PWD" -w "$PWD" --user "$(id -u):$(id -g)" \
    ghcr.io/becklaboratory/pav3:latest batch

Apptainer (or Singularity):

apptainer pull pav3.sif oras://ghcr.io/becklaboratory/pav3-sif:latest

apptainer run pav3.sif batch

The container runs pav3 itself, so arguments follow the image exactly as they would on the command line (batch, --help, and so on).

Both examples give PAV access to the current directory only. If the reference genome or the assemblies are somewhere else, make those locations visible as well: add -v /path:/path for Docker or --bind /path for Apptainer.

Replace latest with a version (e.g. 3.0.0.dev24) to pin a specific release.

Dependencies

Currently, PAV needs minimap2 in the environment where it is run (the containers above already include it). This may change in future releases. All other dependencies are handled by the installer.

Output

PAV will output a VCF file NAME.vcf.gz to the run directory for each sample.

• results/NAME/call_hap: Unmerged variant calls.
  • Includes tables of callable regions in reference ("callable_ref") and query ("callable_qry") coordinates.
• results/NAME/call: Variant calls merged across haplotypes.

All tables are in parquet format.

Configuring PAV for batch runs

To run assemblies in batches ("pav3 batch" command), PAV reads two configuration files:

• pav.json: Points to the reference genome and can be used to set optional parameters.
• assemblies.tsv: A table of input assemblies.

Base config: pav.json

A JSON configuration file, pav.json, configures PAV. Default options are built-in, and the only required option is reference pointing to a reference FASTA file.

Example:

{
  "reference": "/path/to/hg38.no_alt.fa.gz"
}

Assembly table

The assembly table points PAV to input assemblies. It may be in TSV, CSV, Excel, or parquet formats (TSV and CSV may optionally be gzipped). Each assembly has one row in the table.

Columns:

• name: Assembly or sample name.
• hap_*: One column for each assembled haplotype.

Name column

The name column contains the assembly name (or sample name). This column must be present and each row must have a unique value.

Haplotype columns

PAV accepts one or more assembled haplotypes per assembly, each with a column in the table starting with "hap_". Each is a path to an input file for one assembly haplotype.

Common column names are "hap_h1" for haplotype "h1" and "hap_h2" for haplotype "h2". For some assemblies with known parental origins, "hap_pat" and "hap_mat" are commonly used.

There must be at least one haplotype per assembly, and PAV has no limits on the number of haplotypes (i.e. 3 or more are acceptable).

Not all assemblies need to have the same haplotypes. PAV will ignore empty "hap_" values for each assembly where the path is empty. For example, if some assemblies have an "unphased" haplotype and others do not, include the "hap_unphased" column and leave it blank for assemblies that do not have it.

Note that genotypes in the VCF file will have one allele for each haplotype defined for the assembly. For an assembly with haplotypes "h1", "h2", and "unphased", three genotype alleles will be possible (e.g. "1|0|." for a heterozygous variant present in "h1", not present in "h2", and uncallable in "unphased"). The order of genotypes is determined by the order of haplotype columns in the assembly table.

Each "hap_" column contains paths to input files in FASTA, FASTQ, GFA, or FOFN format. FOFN may contain paths to these same file types including other FOFNs (recursive FOFNs are not recommended, but PAV will detect cycles). Multiple files can be input by separating them by semi-colons (i.e. "path/to/file1.fasta.gz;path/to/file2.fasta.gz") and a mix of types is possible.

PAV will be fastest if the input is a single indexed FASTA file, which may be plain text or bgzipped (Samtools bgzip). For all other input including multiple FASTA files or any GFA files for one haplotype, PAV will build a single FASTA file from the input, index it, and use it for variant calling.

Configuration column for global overrides

An optional "config" column can override global configuration parameters per assembly. Global configuration parameters are defined in pav.json or are PAV default values if not defined. Values in this column are semicolon-separated lists of key-value pairs (i.e. "key1=val1;key2=val2"). The "reference" parameter cannot be set in the assembly table.

Suitable genome references

Align against primary assemblies composed of chromosome scaffolds including unplaced contigs and unlocalized contigs if they exist (GRCh/hg references). Do not use references with ALT, PATCH, or DECOY scaffolds. Reference redundancy from these non-primary contigs may increase callset errors.

The GRCh38 HGSVC no-ALT reference for long reads was shared by the HGSVC consortium: https://ftp.1000genomes.ebi.ac.uk/vol1/ftp/data_collections/HGSVC2/technical/reference/20200513_hg38_NoALT/

The T2T-CHM13v2.0 (hs1 on UCSC) is suitable without alteration.

Custom per-sample assemblies containing a single-haplotype or an unphased ("squashed") assembly typically also make a suitable reference as long as they are free of large structural misassemblies and large false duplications. For phased assemblies, choose only one haplotype as a reference.

PAV versions

PAV uses Python package versioning with three fields:

• Major: Major changes or new features.
• Minor: Small changes, but may affect PAV's API or command-line interfaces.
• Patch: Small changes and minor new features. Patch versions do not break API or command-line compatibility, but may add new features.

PAV follows Python's packaging versioning scheme (https://packaging.python.org/en/latest/discussions/versioning/).

PAV may use pre-release versions with a suffix for development releases (".devN"), alpha ("aN"), beta ("bN"), or release-candidate ("rcN") where "N" is an integer greater than 0. For example, "3.0.0.dev1" is a development version, and "3.0.0a1" is an early alpha version, and "3.0.0rc1" is a release candidate, all of which precede the "3.0.0" release and should not be considered production-ready.

Reporting bugs

Please report problems you encounter on the issues page.

Cite PAV

PAV 3 does not yet have a citation. For now, use the citation for previous PAV versions, but check back for updates.

Ebert et al., “Haplotype-Resolved Diverse Human Genomes and Integrated Analysis of Structural Variation,” Science, February 25, 2021, eabf7117, https://doi.org/10.1126/science.abf7117 (PMID: 33632895).

License

PAV is released under the MIT License. See LICENSE.