henhao
Sign in
GenomicsbioRxiv
Preprint

ChlORIS: Chloroplast Orthologs Resource & Identification Suite

Yuhao TongiD, Vanessa R. MarcelinoiD, Robert TurnbulliD, Heroen VerbruggeniD

Abstract

Chloroplast or plastid genomes are essential resources for studying the evolution and diversity of algae and land plants. Although thousands of plastid genomes have been sequenced, their full potential has not been realised; derived resources such as orthogroup databases and reference datasets for metagenomic profiling remain underdeveloped. We present the ChlORIS database to address these problems across all algal phyla. From 2,254 publicly available algal plastid genomes, after dereplication we clustered 2,531 orthogroups from the annotated proteins and selected 496 orthogroups with consistent gene naming, enabling cross-genome comparisons of homologous plastid proteins. We further selected 224 core orthogroups, each containing more than 10 protein sequences, for which we produced score-calibrated hidden Markov models (HMMs), multiple sequence alignments and predicted protein structures. The value of these resources for phylogenomics is demonstrated through a large-scale plastid phylogeny of 859 taxa spanning all major algal lineages. We characterised the protein HMMs by cross-referencing them to Pfam domains and calibrated score cutoffs for reliable detection. The metagenomic database, HMM library, nucleotide and amino acid alignments, predicted structures and protein metadata, cross-linked to UniProt and InterPro (Pfam), are openly available on the ChlORIS website at https://chloris.codeberg.page/.

PDFDOICodeData
Full-text digest ↓

02 /Specification & Classification

Click any tag to browse matching papers
Citations
0
OpenAlex Topics

In-depth summary

✦ Multi-agent verified full-text synthesis

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (11 Aug 2026) · CC BY 4.0

Open plastid orthogroup resource for algae: 496 named orthogroups from 893 genomes, 224 core profile hidden Markov models with calibrated cutoffs, alignments, predicted structures, 859-genome phylogeny, CheckM-compatible marker sets.

Algal organelle databases such as OGDA (Organelle Genome Database for Algae) store genome metadata but not protein families or gene-level evolutionary relationships, and metagenome-assembled genome (MAG) tools such as CheckM lack plastid-specific references, leaving plastid genome reuse to ad hoc pipelines. ChlORIS combines orthogroups, codon and protein alignments, quality-scored HMMs, Pfam annotations, predicted structures and a backbone tree for all algal phyla in one openly licensed resource.

Key results

  1. ShownDereplicating 2,254 GenBank algal plastid genomes at 95% average nucleotide identity (ANI) and dropping unannotated ones left 893 genomes. Their proteins clustered into 2,531 orthogroups: 496 with consistent gene names form the full set, and the 224 of these with >10 sequences form the core set.

    Galah v.0.4.2 reduced 2,254 genomes to 903, and 893 of them had annotations. OrthoHMM v.0.1.0 produced 2,531 orthogroups. Manual curation left out 2,035 orthogroups, which include 1,229 singletons and are mostly uncharacterised hypothetical proteins; they can still be downloaded separately. · Genome preparation; Orthogroup inference and alignments; ChlORIS database contents; Data availability and the ChlORIS website

  2. SuggestedCalibrated noise and trusted bit-score cutoffs removed most off-target hits from the 224 core HMMs. The authors attribute those hits to paralogous plastid proteins that share domains.

    Tested on ca. 116,000 clustered plastid proteins (OrthoHMM assignments as ground truth), hmmsearch at e-value 10^-3 left some outlier HMMs at F1 ≤0.8 with off-target hits, attributed to domain-sharing among paralogs (illustrated by the phycobiliprotein superfamily and psbA/psbD, psbB/psbC). Calibrated cutoffs left only a few proteins at F1 ≤0.85; cutoffs were set and evaluated on the same pool. · Curation of HMMs; Calibrating HMM score cutoffs; Calibrated cutoffs reduce off-target hits

  3. ShownHMM-HMM alignment against Pfam gave 470 of the 496 named orthogroups at least one significant domain hit. This provides functional annotation that does not depend on the original GenBank labels.

    hhalign (HH-suite v.3.3.0) aligned 4,874 Pfam-A HMMs (Pfam r27) to the 496 orthogroup HMMs, and 989 Pfam domains were involved. Cosine similarity of domain profiles was mapped within 9 functional groups (psa, psb, atp, pet, rps, rpl, rpo, phycobilisome, ycf). · Protein structure and Pfam domains; ChlORIS database contents

  4. SuggestedA 51-gene supermatrix from 859 photosynthetic plastid genomes plus 7 cyanobacterial outgroups produced a tree that spans nearly all major primary and secondary plastid lineages. The authors report that phylum placements agree closely with earlier studies.

    The tree has 866 taxa and was built with MAFFT v.7.526 localpair, ClipKIT v.2.7.0 trimming, PhyKIT v.2.1.2 concatenation and IQTREE v2.3.6 (1000 ultrafast bootstraps, MFP+MERGE). 34 non-photosynthetic genomes were pruned. Agreement with earlier studies is stated only in words; no topology comparison or bootstrap summary is reported. · Phylogenetics; Phylogeny of 859 plastid genomes

  5. ClaimedMarker gene sets for 39 monophyletic lineages turn ChlORIS into a CheckM-format database for estimating the completeness and contamination of plastid MAGs. The paper reports no accuracy evaluation of this database.

    The lineages come from the phylogeny. Markers are genes present as a single copy in >95% of a lineage's members, under modified CheckM criteria. The paper refers readers to the separate ChloroScan software description for use with CheckM or binny. · Quality-assessment database for plastid MAGs; Use case examples and recipes

Methods, data and reproducibility

The authors dereplicated GenBank algal plastid genomes, clustered their proteins into orthogroups with OrthoHMM and curated them by gene name. Per orthogroup they built MAFFT protein and codon alignments, profile HMMs with calibrated HMMER cutoffs, Pfam links via HMM-HMM alignment and AlphaFold 3 structures, then built a phylogeny and a CheckM-format database.

  • Plastid genomes from GenBank (land plants, derived plant plastids and sacoglossan sea slug kleptoplasts excluded); 903 after 95% ANI dereplication, 893 with annotations retained · micro- and macroalgae (land plants excluded) · n = 2,254 genomes (893 retained)
  • Clustered plastid proteins used as the reference set for HMM cutoff calibration · micro- and macroalgae · n = ca. 116,000 proteins
  • Cyanobacterial genomes used as phylogenetic outgroups, including the Gloeomargarita lineage · cyanobacteria · n = 7 genomes
  • Pfam-A profile HMMs (Pfam r27) used for domain annotation · n = 4,874 HMMs
Methods & tools
Galah v.0.4.2, OrthoHMM v.0.1.0, MAFFT v.7.526, HMMER v.3.4 (hmmsearch), HH-suite v.3.3.0 (hhalign), Pfam, AlphaFold 3, ClipKIT v.2.7.0, PhyKIT v.2.1.2, IQTREE v2.3.6, CheckM, graft toolkit
Code
https://codeberg.org/chlorishttps://codeberg.org/chloris/graft
Data & accessions
https://chloris.codeberg.page/https://doi.org/10.5281/zenodo.20845450
Software & environment
Tool versions are given: Galah v.0.4.2, OrthoHMM v.0.1.0, MAFFT v.7.526, HMMER v.3.4, HH-suite v.3.3.0, ClipKIT v.2.7.0, PhyKIT v.2.1.2, IQTREE v2.3.6, OrthoFisher v.1.1.2; Pfam r27. No container or requirements file is described.
Reproducibility gaps
The code is said to cover only 'aspects of' the construction workflow. GenBank accessions are listed on the website, not in the text. Predicted structures fall under the AlphaFold 3 terms of use rather than CC0. Hardware and runtime are not reported.
Limitations (5)
  • Reference genomes come mainly from lineages with canonical plastid structure. Non-canonical plastids of dinoflagellates and Cladophorales green algae are not yet included. · stated by the authors
  • Dereplication at 95% ANI merges related species, so diversity within genera is under-represented. · stated by the authors
  • Taxonomy and gene and intron boundaries are taken from GenBank records without re-annotation, so errors in those records carry over into the database. · stated by the authors
  • HMM cutoffs are set and evaluated on the same ca. 116,000 proteins, against the OrthoHMM clustering, with no held-out genomes. The F1 gains may therefore overstate performance on new or divergent genomes. · noted in this summary
  • Neither the CheckM-format database nor the graft toolkit is benchmarked here: there is no test of completeness or contamination accuracy on real or simulated MAGs. · noted in this summary

Who should read:Read this if you work on algal plastid phylogenomics, need curated plastid marker HMMs or alignments, or want to recover plastid MAGs from metagenomes and estimate their completeness.

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISFreudenthal '20Jamy '25Mohanta '20Mu '19Raval '23Li '19
61% semantic matchBioinformatics
Jan A. Freudenthal, Simon Pfaff, Niklas Terhoeven+3 more·bioRxiv·Aug 20, 2020· 15 citations