PGGT

PGGT is a recoverable multi-genome workflow that converts pangenome graph projections into allele, copy-number variation (CNV), and presence/absence variation (PAV) components.

It runs five modules:

1. project annotated genes onto GFA walks and build initial components;
2. filter graph-supported components with DIAMOND and gene structure evidence;
3. score every gene and mark low-score genes as noncoding;
4. regroup singleton genes with reciprocal Miniprot and Pfam evidence;
5. select representative genes and write allele/CNV/PAV component tables and W-only GFA files.

Genes with a total module-3 score below 1.0 remain in the main component table. Their output IDs use ng instead of the terminal gene marker g, for example B73_C05g06442 becomes B73_C05ng06442. The original-to-output ID relationship is recorded in 13.gene_id_mapping.tsv and propagated through modules 4 and 5.

What's new in 1.2.0

• Module 3 scores every gene in every component class using CDS, exon, Pfam, and expression evidence.
• Pfam best hits are selected by the smallest independent E-value, then the largest HMM coverage and domain name as deterministic tie-breakers.
• --previous-pfam-results reuses compatible Pfam calls from an earlier 07.pfam_scores.tsv or PGGT result directory. Reuse is validated by the Pfam HMM fingerprint and protein sequence SHA256; unmatched sequences are scanned normally.
• Low-score gene IDs are marked with ng in module 3 and remain marked in 04.component_cnv and 05.allele_CNV_PAV outputs.

Installation

PGGT requires Linux and Python 3.9 or later.

pip install pggt

The workflow also calls external command-line programs that are not installed by pip:

• bedtools
• diamond
• samtools
• HMMER (hmmscan and hmmpress)
• miniprot

For example, these programs can be prepared with Conda/Bioconda:

conda create -n pggt -c conda-forge -c bioconda \
  python=3.10 bedtools diamond samtools hmmer miniprot
conda activate pggt
pip install pggt

Confirm that the command is available:

pggt --help

Quick start

Run input validation first. --dry-run creates no result files.

pggt \
  --gfa-input pangenome.gfa \
  --genome-dir genomes/ \
  --gff-dir annotations/ \
  --samples species_list.txt \
  --expression-dir expression/ \
  --pfam-hmm Pfam-A.hmm \
  --output-dir pggt_out/ \
  --threads 56 \
  --parallel-jobs 4 \
  --dry-run

After validation succeeds, run the same command without --dry-run.

To reuse Pfam work when rerunning the same species and annotations, add either an earlier PGGT result directory or its compact Pfam table:

pggt [all required arguments] \
  --previous-pfam-results previous_pggt_out/

The previous and current runs must use a Pfam database with the same fingerprint. Reuse is sequence-based, so proteins absent from the previous table or with changed SHA256 values are scanned automatically.

PGGT 1.1 tables do not retain the statistics for every accepted hit. To avoid carrying the 1.1 best-hit direction bug into 1.2, unambiguous no-domain and single-domain legacy rows are reusable; legacy multi-domain rows use an exact shared-cache record when available and otherwise are rescanned. Tables created by PGGT 1.2 retain the corrected best hit and are fully reusable.

The output directory must be new. Do not use a GraphTribe result directory as the PGGT output directory.

Input requirements

Sample list

species_list.txt must contain exactly one sample name per non-empty line and at least two unique samples:

B73
EIL02
EIL31
EIL08
EIL37

Sample names may contain letters, numbers, underscores, dots, and hyphens. The first sample is the reference sample used for representative-gene selection. Every listed sample must occur in the second field of at least one GFA W record.

The same sample names are used to resolve genome, GFF, and expression files. Each sample must resolve to exactly one file of each type.

GFA

--gfa-input accepts either one GFA file or a directory containing exactly one *.gfa file. Use --gfa-name FILE when a directory contains multiple GFA files.

GFA W coordinates are interpreted as 0-based, half-open intervals.

Genome FASTA files

Provide one uncompressed genome FASTA per sample. Recommended names are:

genomes/B73.fa
genomes/EIL02.fa

PGGT first checks <sample>.fa, <sample>.fasta, <sample>.fna, and <sample>.fas. If none exists, it accepts one uniquely matching FASTA whose name contains the sample name.

FASTA sequence names must match the chromosome or sequence names used by the corresponding GFF and GFA walks.

GFF/GFF3 files

Recommended names are:

annotations/B73.renamed.sorted.chr.gff3
annotations/EIL02.renamed.sorted.chr.gff3

PGGT also checks <sample>.gff3 and <sample>.gff, followed by one unique matching GFF file whose name contains the sample name.

The annotations must provide gene, transcript, exon, and CDS relationships. Gene IDs must be consistent with the expression tables and use the supported terminal g plus digits pattern, such as B73_C05g06442; this allows PGGT to mark a noncoding output ID as B73_C05ng06442 without ambiguity. Chromosome names must match the corresponding genome FASTA.

Expression tables

Expression input is required for every sample. Each file must be a tab-separated TSV table. The recommended file name is <sample>.FPKM.tsv; <sample>.expression.tsv is also accepted.

Example:

Gene_ID	leaf_FPKM	root_FPKM
B73_C01g00001	12.50	3.20
B73_C01g00002	0	0.08
B73_C01g00003	0	0

Expression-table rules:

• The header must contain the exact, case-sensitive column name Gene_ID.
• At least one expression column must end exactly with _FPKM.
• Gene IDs must match gene IDs in the corresponding GFF/GFF3.
• FPKM cells must be numeric. Do not write NA, N/A, or other text.
• Empty FPKM cells are interpreted as zero.
• When multiple *_FPKM columns are present, PGGT uses the maximum value for each gene.
• Use one row per gene. If a gene ID occurs more than once, the later row replaces the earlier value.
• Genes absent from the expression table receive the no_expression score.

Extra non-FPKM columns are allowed. PGGT first checks <sample>.FPKM.tsv and <sample>.expression.tsv; otherwise, exactly one *<sample>*.tsv file must exist in the expression directory. Ambiguous matches stop the run.

Pfam database

--pfam-hmm must point to a non-empty Pfam-A.hmm file. PGGT prepares the required HMMER index files in its working area when necessary.

Run control

# Resume a previously interrupted PGGT run
pggt [all original arguments] --resume

# Run only one module
pggt [all required arguments] --only-step 3

# Run a module range
pggt [all required arguments] --from-step 2 --to-step 4

Use the same inputs, output directory, thresholds, thread count, and job count when resuming. Completed modules are validated through their completion markers before being skipped.

Threads and shared caches

--threads is the total CPU budget. --parallel-jobs controls concurrent per-sample work, Pfam shards, Miniprot mappings, and PAF parsing, and must not exceed --threads.

For five samples on a sufficiently large 56-core node, --parallel-jobs 5 allows all five Miniprot target mappings to run concurrently. Values above the sample count can still increase Pfam shard concurrency, but should be benchmarked against available memory and storage bandwidth.

Shared caches are stored under ~/.cache/pggt:

• ~/.cache/pggt/pfam/pfam_cache.sqlite3 caches Pfam results by HMM fingerprint and protein sequence SHA256;
• ~/.cache/pggt/miniprot/ stores Miniprot genome indexes.

Set the PGGT_CACHE_DIR environment variable to use a different cache root.

Main outputs

pggt_out/
├── 01.graph_mapping/
├── 02.component_filter/
│   └── 10.filtered_component_table.txt
├── 03.gene_score/
│   ├── 11.scored_gene_component_table.txt
│   ├── 12.nocoding_gene_scores.tsv
│   ├── 13.gene_id_mapping.tsv
│   └── score_tmp_file/
│       ├── 07.pfam_scores.tsv
│       └── 08.singleton_score.tsv
├── 04.component_cnv/
│   └── 04.orthogroup/
│       └── singleton_orthogroup_component_table.txt
├── 05.allele_CNV_PAV/
│   ├── allele_CNV_PAV_component_table.txt
│   └── W_gfa/
│       └── allele_CNV_PAV.W.gfa
└── 00.run_info/
    └── module_timings.tsv

Each completed module contains artifact_manifest.tsv. Large intermediate DIAMOND, Pfam, and Miniprot files are not published into successful result directories. Failed module workspaces remain under OUTPUT_DIR/.work/ for diagnosis.

Standalone module 5

When a compatible module-4 component table already exists, run module 5 directly:

pggt-module5 \
  --component-table singleton_orthogroup_component_table.txt \
  --samples-file species_list.txt \
  --gff-dir annotations/ \
  --gene-id-map 13.gene_id_mapping.tsv \
  --output-dir module5_out/ \
  --representative-seed 1

--gene-id-map is required when the component table contains module-3 ng IDs but the source GFF still contains the original g IDs.

Command overview

pggt --help
pggt-module5 --help

Maintainer notes

Before a PyPI release, run the regression tests, build both the wheel and source distribution, run twine check dist/*, and verify installation in a clean environment. Update this README whenever command-line options or input requirements change.

Major workflow changes must be developed in a new versioned package directory, with matching updates to pyproject.toml, runtime __version__, this README, PGGT流程原理.md, and CHANGELOG.md. Do not modify a previously published version directory in place.

License

License information will be added before the first public release.