Reassemble chloroplast genomes from precomputed assemblies

ChloroFORGE is a bioinformatics pipeline that identifies chloroplast-derived contigs from existing genome assemblies (e.g., generated with hifiasm or verkko using long reads from Oxford Nanopore or PacBio) and reconstructs the complete chloroplast genome using Flye.

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Table of Contents

• Dependencies
• Installation
• Usage
  • Required Arguments
  • Optional Arguments
• Examples
• Output Structure
• Notes & Tips
• Citation

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Dependencies

ChloroFORGE relies on the following tools (installed automatically via setup.sh):

Tool	Version	Link
minimap2	2.30	https://github.com/lh3/minimap2
Flye	2.9.6	https://github.com/mikolmogorov/Flye
seqkit	2.13.0	https://github.com/shenwei356/seqkit
blastn (BLAST+)	2.17.0	https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/
python3	>=3.7	—

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Installation

git clone https://github.com/usadellab/chloroFORGE.git
cd chloroFORGE
bash setup.sh

The setup script will verify or install all required dependencies and prepare the internal pipeline structure.

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Usage

./chloroFORGE.sh -g GENOME -c CHLOROPLAST -t THREADS -o OUTPUT [OPTIONS]

Required Arguments

Flag	Description
-o	Sample name / project identifier
-g	Absolute path to genome assembly (FASTA)
-c	Absolute path to chloroplast reference genome (FASTA)
-t	Number of threads

You can download a chloroplast reference from a closely related species via NCBI GenBank or RefSeq. Search for your organism and download the chloroplast genome in FASTA format. A useful search strategy is to combine the latin organism name with terms such as "chloroplast" and "complete genome". For example, the following query can help identify suitable reference sequences: ("replace_with_species"[Organism]) AND chloroplast[All Fields] AND "complete genome"[Title] Use this chloroplast genome as an input for -c.

Optional Arguments

Flag	Description	Default
-s	Estimated chloroplast genome size	150k
-l	List of contigs representing chromosomes	none
-x	Target chloroplast contig coverage for Flye	50
-f	Minimum contig overlap for Flye assembly	5000
--allow-lowcov	Allow assembly even if coverage is below target	false

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Examples

Basic run

./chloroFORGE.sh \
  -g genome.fasta \
  -c chloroplast_ref.fasta \
  -t 16 \
  -o sample01

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High coverage + custom overlap

For samples with large unanchored contigs, increasing coverage (-x) and adjusting the minimum overlap (-f) can improve assembly quality.

./chloroFORGE.sh \
  -g genome.fasta \
  -c chloroplast_ref.fasta \
  -t 16 \
  -o sample01 \
  -x 80 \
  -f 7000

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Low coverage assembly

If coverage is consistently low, you can either reduce -x and rerun, or use --allow-lowcov to bypass the coverage check entirely.

Warning: Results from low-coverage assemblies should be inspected carefully, as they may be incomplete or incorrect.

./chloroFORGE.sh \
  -g genome.fasta \
  -c chloroplast_ref.fasta \
  -t 16 \
  -o sample01 \
  --allow-lowcov

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Output Structure

After a successful run, the output directory will have the following structure:

sample01/
├── blast_hits.tsv                          # Raw BLAST hits
├── cp_contigs.txt                          # Contig IDs identified as chloroplastic
├── cp_contigs.fasta                        # Extracted chloroplast contigs
├── flye_cp_out/                            # Flye output directory
├── results_cp/
│   ├── edges.fa                            # Flye assembly graph edges
│   ├── edges_depth                         # Per-edge depth file
│   └── chloroplast_final_assembly/
│       └── sample01_chloroplast.fasta      # Final oriented chloroplast assembly
└── final_genome_sample01.fasta             # Final genome (non-cp sequences + chloroplast)

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Notes & Tips

• Assembly validation: The pipeline stops if the final assembly length deviates more than ±10% from the reference chloroplast length. This is a sanity check — inspect your inputs if this triggers.
• Coverage tuning: The default target coverage of 50x works well in most cases. If assembly fails, try lowering -x before resorting to --allow-lowcov.
• Overlap tuning: The default minimum overlap (-f 5000) can be decreased for fragmented inputs or increased for better-resolved assemblies.
• Reference genome: Always use a chloroplast reference from a closely related species for best BLAST sensitivity.

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Citation

If you use ChloroFORGE in your research, please cite this repository:

ChloroFORGE – https://github.com/usadellab/ChloroFORGE