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https://github.com/lh3/pangene

Constructing a pangenome gene graph
https://github.com/lh3/pangene

bioinformatics pangenome

Last synced: 3 months ago
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Constructing a pangenome gene graph

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README 

        
## Getting Started

```sh

# Check prebuilt graphs at https://pangene.bioinweb.org



# Install pangene

git clone https://github.com/lh3/pangene

cd pangene && make



# Alternatively, download the precompiled binaries for arm-mac and x64-linux

curl -L https://github.com/lh3/pangene/releases/download/v1.1/pangene-1.1-bin.tar.bz2|tar jxf -



# The C4 example with provided alignment

./pangene test/C4/*.paf.gz > C4.gfa        # generate the graph

k8 pangene.js call C4.gfa > C4.bubble.txt  # identify bubbles



# Deploy the GFA server on the C4 example; require pangene-1.1-bin

cd pangene-1.1-bin                         # run gfa-server in this directory

bin_arm64-mac/gfa-server /path/to/C4.gfa   # or use bin_linux-x64 on x64-linux

# open "http://127.0.0.1:8000/view?gene=C4A,C4B" in a web browser



# Deploy the GFA server on the human graph

bin_arm64-mac/gfa-server -d html data/*.gfa.gz 2> server.log &

# open "http://127.0.0.1:8000" in a web browser



# Align proteins to each genome (general use cases; no example data)

miniprot --outs=0.97 --no-cs -Iut16 genome1.fna proteins.faa > genome1.paf

miniprot --outs=0.97 --no-cs -Iut16 genome2.fna proteins.faa > genome2.paf



# Construct a pangene graph

pangene genome1.paf genome2.paf > graph.gfa



# Check manpage

man ./pangene.1

```



## Table of Contents



- [Getting Started](#started)

- [Introduction](#intro)

- [Graph Construction](#build)

  - [Preparing a protein set](#prep-aa)

  - [Aligning proteins to genomes](#align-aa)

  - [Constructing a pangene graph](#build-graph)

  - [Analyzing a graph](#analyze)

- [Graph Visualization](#visual)

- [Citation](#cite)

- [Limitations](#limit)



## Introduction



Pangene is a command-line tool to construct a pangenome gene graph. In this

graph, a node repsents a marker gene and an edge between two genes indicates

their genomic adjaceny on input genomes. Pangene takes the [miniprot][mp]

alignment between a protein set and multiple genomes and produces a graph in 

the GFA format. It attempts to reduce the redundancy in the input proteins and

filter spurious alignments while preserving close but non-identical paralogs.

The output graph can be visualized in generic GFA viewers such as

[BandageNG][bandage] or via a [web interface](#visual). Users can explore local

human subgraphs at a [public server][server]. Prebuilt pangene graphs can be

found at [DOI:10.5281/zenodo.8118576][zenodo].



## Graph Construction



Pangene takes a list of protein-to-genome alignment as input. To generate

these alignments, you need to align the same set of proteins to multiple

genomes. How to choose the protein set can be tricky.



### Preparing a protein set



For constructing a human pangene graph, the simplest choice is to use annotated

genes in GRCh38. It is highly recommended to name a protein sequence like

`RGPD6:ENSP00000512633.1` where `RGPD6` is the gene name and

`ENSP00000512633.1` is the unique protein identifier. In the output GFA, nodes

are named after genes, so you would want to use human-readable gene names for

visualization later. You may use the following command line to extract protein

sequences from Ensembl or GenCode annotation:

```sh

k8 pangene.js getaa gene-anno.gtf protein-seq.faa > proteins.faa

```



With pangene, different isoforms or diverged alleles of the same gene can be

present in the protein set, though in practice, we find selecting the canonical

isoform per gene tends to give a cleaner graph probably possibly due to

annotation errors among rare isoforms. For the GenCode annotation, use `getaa

-c` to extract canonical isoforms only.



For a new species without good gene annotation, you may use protein annotations

from a closely related species. You may pool proteins from multiple closely

related species as well. Pangene aims to work with such input but this use case

has not been thoroughly carefully evaluated. Given a bacteria pangenome of the

same species, you may predict genes with existing tools, cluster them with

CD-HIT or MMseqs2 and feed the representative protein in each cluster to

pangene.



### Aligning proteins to genomes



Pangene currently only works with miniprot's PAF output. We usually use the

following command line:

```sh

miniprot --outs=0.97 --no-cs -Iut16 genomeX.fna proteins.faa > genomeX.paf

```

For bacterial data, add `-S` to disable splicing.



### Constructing a pangene graph



The following command-line constructs a pangene graph

```sh

pangene *.paf > graph.gfa

```

If the output graph is cluttered in the Bandage viewer, you may add option

`-a2` to filter out edges supported by a single genome. By default, pangene

filters out genes occurring in less than 5% of the genomes after deredundancy.

If you want to retain low-frequency genes, add `-p0` to disable the filter.



### Analyzing a graph



The GFA file is the master output. You can extract various information from

this file. You may find local gene-level variations with

```sh

k8 pangene.js call graph.gfa > bubble.txt

```

or get the presence/absence of each gene with

```sh

k8 pangene.js gfa2matrix graph.gfa > gene_presence_absence.Rtab

```



## Graph Visualization



You can look at the entire graph in the Bandage GFA viewer. If you are

interested in a particular gene, it is best to set up gfa-server which is part

of [gfatools][gfatools]. [Here][server] is a public server for human genes.

You can deploy this server on your machine with

```sh

curl -L https://github.com/lh3/pangene/releases/download/v1.1/pangene-1.1-bin.tar.bz2|tar jxf -

cd pangene-1.1-bin

bin_arm64-mac/gfa-server -d html data/*.gfa.gz 2> server.log # for Mac

```

Then you can open link `http://127.0.0.1:8000/` in your browser, type gene

names and visualize a local subgraph around the desired genes.



## Citation



If you use pangene in your work, please consider to cite:



> H Li, M Marin, MR Farhat (2024) Exploring gene content with pangenome gene graphs,

> arXiv:2402.16185



## Limitations



* Pangene only works with [miniprot][mp]'s PAF output.



* In the output graph, arcs on W-lines may be absent from L-lines.



[mp]: https://github.com/lh3/miniprot

[bandage]: https://github.com/asl/BandageNG

[gfatools]: https://github.com/lh3/gfatools

[gfaview]: https://lh3.github.io/gfatools/

[panaroo]: https://github.com/gtonkinhill/panaroo

[asub]: https://github.com/lh3/asub

[zenodo]: https://doi.org/10.5281/zenodo.8118576

[server]: https://pangene.bioinweb.org