The assessment by Brézellec (2024) of the quality of HHpred-based SARS-CoV-2 protein annotations against the traditional Pfam annotations is highly justified and valuable. HHpred’s ability to detect remote homologies offers an expanded view of viral protein similarities, potentially uncovering subtle functional mimicries that Pfam may miss due to its sensitivity limitations when dealing with divergent sequences. However, the accuracy and specificity of HHpred results can be compromised by false positives, especially when dealing with complex viral proteins that feature transmembrane or low-complexity regions prone to spurious matches.

To address this, the author made a thoughtful decision to implement a multi-step validation protocol. This approach included establishing progressively lower probability thresholds to capture weaker but biologically plausible hits, and organizing hits into “families” of similarly located alignments to validate the robustness of matches. They also cross-verified results by running SARS-CoV-2 protein queries against non-human proteomes (plants, fruit flies, bacteria, and archaea), allowing them to discern between biologically meaningful matches and potentially random alignments. By adding manual verification with InterPro domain annotations, the authors took additional steps to ensure that identified similarities were not only statistically significant but also biologically relevant.

This rigorous validation strategy adds a layer of reliability to HHpred results, demonstrating an effective maximization of sensitivity while maintaining specificity. This approach yielded biologically intriguing and previously undocumented similarities, such as between the Spike-prominin and ORF3a-GPCR, underscoring the quality and depth of the annotation process. These findings highlight a pathway for further experimental validation and illustrate the potential of HHpred to contribute high-quality insights when applied with careful quality control measures.

In summary, the decision to adopt HHpred (Gabler et al. 2020) and enhance its outputs with a robust quality validation process not only improved the depth of SARS-CoV-2 protein annotations but also established a high standard for future viral annotation projects, striking an effective balance between discovery potential and annotation quality​. The authors have conducted a study that is methodologically rigorous, well-detailed, and highly pertinent to the field. This work stands as a significant contribution to the scientific community, providing resources and insights that are likely to guide future research in this area. 

              
References

Brézellec, P (2024) Re-annotation of SARS-CoV-2 proteins using an HHpred-based approach opens new opportunities for a better understanding of this virus. bioRxiv, ver. 3 peer-reviewed and recommended by PCI Genomics. https://doi.org/10.1101/2023.06.06.543855

Gabler F, Nam S-Z, Till S, Mirdita M, Steinegger M, Söding J, Lupas AN, Alva V (2020) Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Current Protocols in Bioinformatics, 72, e108. https://doi.org/10.1002/cpbi.108

The first time I heard of ”onagres” in French was when I was a teenager, through the books of Pierre Bordage as fantastic monsters, or through historical games as Roman siege weapons (onagers). At this time, I was far from imagining that “onagre” also refers to a very large flowering plant family, as it is the French term for evening primroses.

In this family, the genus Ludwigia comprises species that are invasive (resembling in that way the ancient armies using onagers to invade cities) in aquatic environments, degrading ecosystems already fragilized by human activities. To counteract this phenomenon, it is of high importance to understand their propagation of these species. However, the knowledge about their genetics and diversity is very scarce, and thus tracking their dispersal using genetic information is complicated, and in fact almost impossible.

Barloy-Hubler et al. (2024) proposed in the present manuscript a new set of chloroplastic genomes from two of these species, Ludwigia grandiflora subsp. hexapetala and Ludwigia peploides subsp. montevidensis, and compared them to the published chloroplastic genome of Ludwigia octovalis. They explored the possibility of assembling these genomes relying solely on short reads and showed that long reads were necessary to obtain an almost complete assembly for these plastid genomes. In addition, through this approach, they detected two haplotypes in Ludwigia grandiflora subsp. hexapetala as compared to one in a short-read assembly. This highlights the need for long reads data to assess the structure and diversity of chloroplastic genomes. The authors were also able to clarify the phylogeny of the genus Ludwigia. Finally, they identified multiple potential single nucleotide polymorphisms and simple sequence repeats for future evaluation of diversity and dispersal of those invasive species.

This analysis, while appearing more technical than biological at first glance, is in fact of high importance for the understanding of ecology and preservation of fragile ecosystems, such as the European watersheds. Indeed, new scientific results and insights are generally linked to a reevaluation of previously analyzed data or samples through new technologies, and this paper is a quite clever example of that matter.

References

Barloy-Hubler F, Gac A-LL, Boury C, Guichoux E, Barloy D (2024) Sequencing, de novo assembly of Ludwigia plastomes, and comparative analysis within the Onagraceae family. bioRxiv, ver. 5 peer-reviewed and recommended by PCI Genomics. https://doi.org/10.1101/2023.10.20.563230

Bordage, P (1993) Les Guerriers du Silence, L'Atalante, ISBN 9782905158697

Any multicellular organism is a molecular mosaic with some somatic mutations accumulated between cell lineages. Big long-lived trees have nourished this imaginary of a somatic mosaic tree, from the observation of spectacular phenotypic mosaics and also because somatic mutations are expected to potentially be passed on to gametes in plants (review in Schoen and Schultz 2019). The lower cost of genome sequencing now offers the opportunity to tackle the issue and identify somatic mutations in trees.

However, when it comes to characterizing this somatic mosaic from genome sequences, things become much more difficult than one would think in the first place. What separates cell lineages ontogenetically, in cell division number, or in time? How to sample clonal cell populations? How do somatic mutations distribute in a population of cells in an organ or an organ sample? Should they be fixed heterozygotes in the sample of cells sequenced or be polymorphic? Do we indeed expect somatic mutations to be fixed? How should we identify and count somatic mutations?

To date, the detection of somatic mutations has mostly been done with a single variant caller in a given study, and we have little perspective on how different callers provide similar or different results. Some studies have used standard SNP callers that assumed a somatic mutation is fixed at the heterozygous state in the sample of cells, with an expected allele coverage ratio of 0.5, and less have used cancer callers, designed to detect mutations in a fraction of the cells in the sample. However, standard SNP callers detect mutations that deviate from a balanced allelic coverage, and different cancer callers can have different characteristics that should affect their outcomes.

In order to tackle these issues, Schmitt et al. (2022) conducted an extensive simulation analysis to compare different variant callers. Then, they reanalyzed two large published datasets on pedunculate oak, Quercus robur.  The analysis of in silico somatic mutations allowed the authors to evaluate the performance of different variant callers as a function of the allelic fraction of somatic mutations and the sequencing depth. They found one of the seven callers to provide better and more robust calls for a broad set of allelic fractions and sequencing depths. The reanalysis of published datasets in oaks with the most effective cancer caller of the in silico analysis allowed them to identify numerous low-frequency mutations that were missed in the original studies.

I recommend the study of Schmitt et al. (2022) first because it shows the benefit of using cancer callers in the study of somatic mutations, whatever the allelic fraction you are interested in at the end. You can select fixed heterozygotes if this is your ultimate target, but cancer callers allow you to have in addition a valuable overview of the allelic fractions of somatic mutations in your sample, and most do as well as SNP callers for fixed heterozygous mutations. In addition, Schmitt et al. (2022) provide the pipelines that allow investigating in silico data that should correspond to a given study design, encouraging to compare different variant callers rather than arbitrarily going with only one. We can anticipate that the study of somatic mutations in non-model species will increasingly attract attention now that multiple tissues of the same individual can be sequenced at low cost, and the study of Schmitt et al. (2022) paves the way for questioning and choosing the best variant caller for the question one wants to address.

References

Schoen DJ, Schultz ST (2019) Somatic Mutation and Evolution in Plants. Annual Review of Ecology, Evolution, and Systematics, 50, 49–73. https://doi.org/10.1146/annurev-ecolsys-110218-024955

Schmitt S, Leroy T, Heuertz M, Tysklind N (2022) Somatic mutation detection: a critical evaluation through simulations and reanalyses in oaks. bioRxiv, 2021.10.11.462798. ver. 4 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2021.10.11.462798

The wide adoption of high-throughput sequencing technologies has uncovered an astonishing diversity of viruses in most biosphere habitats. Among them, single-stranded DNA viruses are prevalent, infecting diverse hosts from all three domains of life (Malathi et al. 2014) with some species being highly pathogenic to animals or plants.

Sequencing of single-stranded DNA viruses requires a specific approach that usually leads to their over-representation compared to double-stranded DNA. The article from Billaud et al. (2024) addresses this challenge. It presents a novel and efficient method for converting single-stranded DNA to double-stranded DNA using T7 DNA polymerase before high-throughput virome sequencing. It compares this new method with the Phi29 polymerase method, demonstrating its advantages in the representation and accuracy of viral DNA content in well-defined synthetic phage mixtures and complex human virome samples from the stool. This T7 DNA polymerase treatment significantly improved the richness and abundance of the Microviridae fraction in their samples, suggesting a more comprehensive representation of viral diversity.

The article presents a compelling case for testing and adopting the T7 DNA polymerase methodology in preparing virome samples for shotgun sequencing. This novel approach, supported by comparative analysis with existing methodologies, represents a valuable contribution to metagenomics for characterizing virome diversity.

References

Billaud M, Theodorou I, Lamy-Besnier Q, Shah SA, Lecointe F, Sordi LD, Paepe MD, Petit M-A (2024) T7 DNA polymerase treatment improves quantitative sequencing of both double-stranded and single-stranded DNA viruses. bioRxiv, ver. 4 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2022.12.12.520144

Malathi VG, Renuka Devi P. (2019) ssDNA viruses: key players in global virome. Virus disease. 30: 3–12. https://doi.org/10.1007/s13337-019-00519-4 

The European Reference Genome Atlas (ERGA) (Mc Cartney et al, 2024, Mazzoni et al, 2023) demonstrates the collaborative spirit and intellectual abilities of researchers from 33 European countries. This ambitious project, which is part of the Earth BioGenome Project (Lewin et al., 2018) Phase II, has embarked on an unprecedented mission: to decipher the genetic makeup of 150,000 species over a span of four years. At the heart of ERGA is a decentralized pilot infrastructure specifically built to assist the production of high-quality reference genomes. This structure acts as a scaffold for the massive task of genome sequencing, giving the necessary framework to manage the complexity of genomic research. The research paper under consideration offers a comprehensive narrative of ERGA's evolution, outlining both successes and challenges encountered along the road. 

One of the most significant issues addressed in the manuscript is the equitable distribution of resources and expertise among participating laboratories and countries. In a project of this magnitude, it is critical to leverage the pooled talents and capacities of researchers from across Europe. ERGA's pan-European network promotes communications and collaboration, creating an environment in which knowledge flows freely and barriers are overcome. This adoption of strong coordination and communication tactics will be essential to ERGA's success. Scientific collaboration depends on efficient communication channels because they allow researchers to share resources, collaborate on new initiatives, and exchange ideas. Through a diverse range of gatherings, courses, and virtual discussion boards, ERGA fosters an environment of transparency and cooperation among members, enabling scientists to overcome challenges and make significant discoveries. The importance ERGA places on training and information transfer programmes is a pillar of its strategy. Understanding the importance of capacity development, ERGA invests in providing researchers with the knowledge and abilities necessary for effectively navigating the complicated terrain of genomic research. A wide range of subjects are covered in training programmes (Larivière et al. 2023), from sample preparation and collection to data processing methods and sequencing technology. Through the development of a group of highly qualified experts, ERGA creates the foundation for continued advancement and creativity in the genomics sector.

This manuscript also covers in detail the technological workflows and sequencing techniques used in ERGA's pilot infrastructure. With the aid of cutting-edge sequencing technologies based on both long-read and short-read sequencing, they are working to unravel the complex structure of the genetic code with a level of accuracy and precision never before possible. To guarantee the accuracy of genetic data and prevent mistakes and flaws that can jeopardize the findings' integrity, quality control methods are put in place. Despite having a focus on genome sequencing due to its technological complexities, ERGA also remains firm in its dedication to metadata collection and sample validation. Metadata serves as a critical link between raw genetic data and useful scientific insights, giving necessary context and allowing researchers to draw practical findings from their investigations. Sample validation approaches improve the reliability and reproducibility of the results, providing users confidence in the quality of the genetic data provided by ERGA.​

Looking ahead, ERGA envisions its decentralized infrastructure serving as a model for global collaborative research efforts. By embracing diversity, encouraging cooperation, and pushing for open access to data and resources, ERGA hopes to catalyze scientific discovery and generate positive change in the field of biodiversity genomics. ERGA aims to promote a more equitable and sustainable future for all by ongoing interaction with stakeholders, intensive outreach and education activities, and policy change advocacy. In addition to its immediate goals, ERGA considers the long-term implications of its work. As genomic technology progresses, the potential application of high-quality reference genomes will continue to grow. From informing conservation efforts and illuminating evolutionary histories to revolutionizing healthcare and agriculture, it is likely that ERGA's contributions will have far-reaching consequences for people and the planet as a whole.​

Furthermore, ERGA understands the importance of interdisciplinary collaboration in addressing the difficult challenges of the twenty-first century. ERGA aims to integrate genetic research into larger initiatives to promote sustainability and biodiversity conservation by forming relationships with stakeholders from other areas, such as policymakers, conservationists, and indigenous groups. Through shared knowledge and community action, ERGA seeks to create a future in which mankind coexists peacefully with the natural world, guided by a thorough grasp of its genetic legacy and ecological interconnectivity.

Finally, the manuscript exemplifies ERGA's collaborative ambitions and achievements, capturing the spirit of creativity and collaboration that defines this ground-breaking effort. As ERGA continues to push the boundaries of genetic research, it remains dedicated to scientific excellence, inclusivity, and the quest of knowledge for the benefit of society. I wholeheartedly recommend the publication of this groundbreaking initiative, offering my enthusiastic endorsement for its valuable contribution to the scientific community.​​

References
Larivière, D., Abueg, L., Brajuka, N. et al. (2024). Scalable, accessible and reproducible reference genome assembly and evaluation in Galaxy. Nature Biotechnology 42, 367-370. https://doi.org/10.1038/s41587-023-02100-3

Lewin, H. A., Robinson, G. E., Kress, W. J., Baker, W. J., Coddington, J., Crandall, K. A., Durbin, R., Edwards, S. V., Forest, F., Gilbert, M. T. P., Goldstein, M. M., Grigoriev, I. V., Hackett, K. J., Haussler, D., Jarvis, E. D., Johnson, W. E., Patrinos, A., Richards, S., Castilla-Rubio, J. C., … Zhang, G. (2018). Earth BioGenome Project: Sequencing life for the future of life. Proceedings of the National Academy of Sciences, 115(17), 4325–4333. https://doi.org/10.1073/pnas.1720115115

Mazzoni, C. J., Claudio, C.i, Waterhouse, R. M. (2023). Biodiversity: an atlas of European reference genomes. Nature 619 : 252-252. https://doi.org/10.1038/d41586-023-02229-w

Mc Cartney, A. M., Formenti, G., Mouton, A., Panis, D. de, Marins, L. S., Leitão, H. G., Diedericks, G., Kirangwa, J., Morselli, M., Salces-Ortiz, J., Escudero, N., Iannucci, A., Natali, C., Svardal, H., Fernández, R., Pooter, T. de, Joris, G., Strazisar, M., Wood, J., … Mazzoni, C. J. (2024). The European Reference Genome Atlas: piloting a decentralised approach to equitable biodiversity genomics. bioRxiv, ver. 4 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2023.09.25.559365​