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16 Dec 2022
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Toeholder: a Software for Automated Design and In Silico Validation of Toehold Riboswitches

A novel approach for engineering biological systems by interfacing computer science with synthetic biology

Recommended by based on reviews by Wim Wranken and 1 anonymous reviewer

Biological systems depend on finely tuned interactions of their components. Thus, regulating these components is critical for the system's functionality. In prokaryotic cells, riboswitches are regulatory elements controlling transcription or translation. Riboswitches are RNA molecules that are usually located in the 5′-untranslated region of protein-coding genes. They generate secondary structures leading to the regulation of the expression of the downstream protein-coding gene (Kavita and Breaker, 2022). Riboswitches are very versatile and can bind a wide range of small molecules; in many cases, these are metabolic byproducts from the gene’s enzymatic or signaling pathway. Their versatility and abundance in many species make them attractive for synthetic biological circuits. One class that has been drawing the attention of synthetic biologists is toehold switches (Ekdahl et al., 2022; Green et al., 2014). These are single-stranded RNA molecules harboring the necessary elements for translation initiation of the downstream gene: a ribosome-binding site and a start codon. Conformation change of toehold switches is triggered by an RNA molecule, which enables translation.

To exploit the most out of toehold switches, automation of their design would be highly advantageous. Cisneros and colleagues (Cisneros et al., 2022) developed a tool, “Toeholder”, that automates the design of toehold switches and performs in silico tests to select switch candidates for a target gene. Toeholder is an open-source tool that provides a comprehensive and automated workflow for the design of toehold switches. While web tools have been developed for designing toehold switches (To et al., 2018), Toeholder represents an intriguing approach to engineering biological systems by coupling synthetic biology with computational biology. Using molecular dynamics simulations, it identified the positions in the toehold switch where hydrogen bonds fluctuate the most. Identifying these regions holds great potential for modifications when refining the design of the riboswitches. To be effective, toehold switches should provide a strong ON signal and a weak OFF signal in the presence or the absence of a target, respectively. Toeholder nicely ranks the candidate toehold switches based on experimental evidence that correlates with toehold performance (based on good ON/OFF ratios).

Riboswitches are highly appealing for a broad range of applications, including pharmaceutical and medical purposes (Blount and Breaker, 2006; Giarimoglou et al., 2022; Tickner and Farzan, 2021), thanks to their adaptability and inexpensiveness. The Toeholder tool developed by Cisneros and colleagues is expected to promote the implementation of toehold switches into these various applications.


Blount KF, Breaker RR (2006) Riboswitches as antibacterial drug targets. Nature Biotechnology, 24, 1558–1564.

Cisneros AF, Rouleau FD, Bautista C, Lemieux P, Dumont-Leblond N, ULaval 2019 T iGEM (2022) Toeholder: a Software for Automated Design and In Silico Validation of Toehold Riboswitches. bioRxiv, 2021.11.09.467922, ver. 3 peer-reviewed and recommended by Peer Community in Genomics.

Ekdahl AM, Rojano-Nisimura AM, Contreras LM (2022) Engineering Toehold-Mediated Switches for Native RNA Detection and Regulation in Bacteria. Journal of Molecular Biology, 434, 167689.

Giarimoglou N, Kouvela A, Maniatis A, Papakyriakou A, Zhang J, Stamatopoulou V, Stathopoulos C (2022) A Riboswitch-Driven Era of New Antibacterials. Antibiotics, 11, 1243.

Green AA, Silver PA, Collins JJ, Yin P (2014) Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell, 159, 925–939.

Kavita K, Breaker RR (2022) Discovering riboswitches: the past and the future. Trends in Biochemical Sciences.

Tickner ZJ, Farzan M (2021) Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors. Pharmaceuticals, 14, 554.

To AC-Y, Chu DH-T, Wang AR, Li FC-Y, Chiu AW-O, Gao DY, Choi CHJ, Kong S-K, Chan T-F, Chan K-M, Yip KY (2018) A comprehensive web tool for toehold switch design. Bioinformatics, 34, 2862–2864.

Toeholder: a Software for Automated Design and In Silico Validation of Toehold RiboswitchesAngel F. Cisneros, François D. Rouleau, Carla Bautista, Pascale Lemieux, Nathan Dumont-Leblond<p>Abstract:&nbsp;Synthetic biology aims to engineer biological circuits, which often involve gene expression. A particularly promising group of regulatory elements are riboswitches because of their versatility with respect to their targets, but e...BioinformaticsSahar Melamed2022-02-16 14:40:13 View
06 Apr 2021
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Evidence for shared ancestry between Actinobacteria and Firmicutes bacteriophages

Viruses of bacteria: phages evolution across phylum boundaries

Recommended by based on reviews by 3 anonymous reviewers

Bacteria and phages have coexisted and coevolved for a long time. Phages are bacteria-infecting viruses, with a symbiotic status sensu lato, meaning they can be pathogenic, commensal or mutualistic. Thus, the association between bacteria phages has probably played a key role in the high adaptability of bacteria to most - if not all – of Earth’s ecosystems, including other living organisms (such as eukaryotes), and also regulate bacterial community size (for instance during bacterial blooms). 

As genetic entities, phages are submitted to mutations and natural selection, which changes their DNA sequence. Therefore, comparative genomic analyses of contemporary phages can be useful to understand their evolutionary dynamics. International initiatives such as SEA-PHAGES have started to tackle the issue of history of phage-bacteria interactions and to describe the dynamics of the co-evolution between bacterial hosts and their associated viruses. Indeed, the understanding of this cross-talk has many potential implications in terms of health and agriculture, among others.

The work of Koert et al. (2021) deals with one of the largest groups of bacteria (Actinobacteria), which are Gram-positive bacteria mainly found in soil and water. Some soil-born Actinobacteria develop filamentous structures reminiscent of the mycelium of eukaryotic fungi. In this study, the authors focused on the Streptomyces clade, a large genus of Actinobacteria colonized by phages known for their high level of genetic diversity.

The authors tested the hypothesis that large exchanges of genetic material occurred between Streptomyces and diverse phages associated with bacterial hosts. Using public datasets, their comparative phylogenomic analyses identified a new cluster among Actinobacteria–infecting phages closely related to phages of Firmicutes. Moreover, the GC content and codon-usage biases of this group of phages of Actinobacteria are similar to those of Firmicutes. 

This work demonstrates for the first time the transfer of a bacteriophage lineage from one bacterial phylum to another one. The results presented here suggest that the age of the described transfer is probably recent since several genomic characteristics of the phage are not fully adapted to their new hosts. However, the frequency of such transfer events remains an open question. If frequent, such exchanges would mean that pools of bacteriophages are regularly fueled by genetic material coming from external sources, which would have important implications for the co-evolutionary dynamics of phages and bacteria.


Koert, M., López-Pérez, J., Courtney Mattson, C., Caruso, S. and Erill, I. (2021) Evidence for shared ancestry between Actinobacteria and Firmicutes bacteriophages. bioRxiv, 842583, version 5 peer-reviewed and recommended by Peer community in Genomics. doi: 

Evidence for shared ancestry between Actinobacteria and Firmicutes bacteriophagesMatthew Koert, Júlia López-Pérez, Courtney Mattson, Steven M. Caruso, Ivan Erill<p>Bacteriophages typically infect a small set of related bacterial strains. The transfer of bacteriophages between more distant clades of bacteria has often been postulated, but remains mostly unaddressed. In this work we leverage the sequencing ...Evolutionary genomicsDenis Tagu2019-12-10 15:26:31 View
24 Sep 2020
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A rapid and simple method for assessing and representing genome sequence relatedness

A quick alternative method for resolving bacterial taxonomy using short identical DNA sequences in genomes or metagenomes

Recommended by based on reviews by Gavin Douglas and 1 anonymous reviewer

The bacterial species problem can be summarized as follows: bacteria recombine too little, and yet too much (Shapiro 2019).
Too little in the sense that recombination is not obligately coupled with reproduction, as in sexual eukaryotes. So the Biological Species Concept (BSC) of reproductive isolation does not strictly apply to clonally reproducing organisms like bacteria. Too much in the sense that genetic exchange can occur promiscuously across species (or even Domains), potentially obscuring species boundaries.
In parallel to such theoretical considerations, several research groups have taken more pragmatic approaches to defining bacterial species based on sequence similarity cutoffs, such as genome-wide average nucleotide identity (ANI). At a cutoff above 95% ANI, genomes are considered to come from the same species. While this cutoff may appear arbitrary, a discontinuity around 95% in the distribution of ANI values has been argued to provide a 'natural' cutoff (Jain et al. 2018). This discontinuity has been criticized as being an artefact of various biases in genome databases (Murray, Gao, and Wu 2020), but appears to be a general feature of relatively unbiased metagenome-assembled genomes as well (Olm et al. 2020). The 95% cutoff has been suggested to represent a barrier to homologous recombination (Olm et al. 2020), although clusters of genetic exchange consistent with BSC-like species are observed at much finer identity cutoffs (Shapiro 2019; Arevalo et al. 2019).
Although 95% ANI is the most widely used genomic standard for species delimitation, it is by no means the only plausible approach. In particular, tracts of identical DNA provide evidence for recent genetic exchange, which in turn helps define BSC-like clusters of genomes (Arevalo et al. 2019). In this spirit, Briand et al. (2020) introduce a genome-clustering method based on the number of shared identical DNA sequences of length k (or k-mers). Using a test dataset of Pseudomonas genomes, they find that 95% ANI corresponds to approximately 50% of shared 15-mers. Applying this cutoff yields 350 Pseudomonas species, whereas the current taxonomy only includes 207 recognized species. To determine whether splitting the genus into a greater number of species is at all useful, they compare their new classification scheme to the traditional one in terms of the ability to taxonomically classify metagenomic sequencing reads from three Pseudomonas-rich environments. In all cases, the new scheme (termed K-IS for "Kinship relationships Identification with Shared k-mers") yielded a higher number of classified reads, with an average improvement of 1.4-fold. This is important because increasing the number of genome sequences in a reference database – without consistent taxonomic annotation of these genomes – paradoxically leads to fewer classified metagenomic reads. Thus a rapid, automated taxonomy such as the one proposed here offers an opportunity to more fully harness the information from metagenomes.
KI-S is also fast to run, so it is feasible to test several values of k and quickly visualize the clustering using an interactive, zoomable circle-packing display (that resembles a cross-section of densely packed, three-dimensional dendrogram). This interface allows the rapid flagging of misidentified species, or understudied species with few sequenced representatives as targets for future study. Hopefully these initial Pseudomonas results will inspire future studies to apply the method to additional taxa, and to further characterize the relationship between ANI and shared identical k-mers. Ultimately, I hope that such investigations will resolve the issue of whether or not there is a 'natural' discontinuity for bacterial species, and what evolutionary forces maintain this cutoff.


Arevalo P, VanInsberghe D, Elsherbini J, Gore J, Polz MF (2019) A Reverse Ecology Approach Based on a Biological Definition of Microbial Populations. Cell, 178, 820-834.e14.
Briand M, Bouzid M, Hunault G, Legeay M, Saux MF-L, Barret M (2020) A rapid and simple method for assessing and representing genome sequence relatedness. bioRxiv, 569640, ver. 5 peer-reveiwed and recommended by PCI Genomics.
Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nature Communications, 9, 5114.
Murray CS, Gao Y, Wu M (2020) There is no evidence of a universal genetic boundary among microbial species. bioRxiv, 2020.07.27.223511.
Olm MR, Crits-Christoph A, Diamond S, Lavy A, Carnevali PBM, Banfield JF (2020) Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries. mSystems, 5.
Shapiro BJ (2019) What Microbial Population Genomics Has Taught Us About Speciation. In: Population Genomics: Microorganisms Population Genomics. (eds Polz MF, Rajora OP), pp. 31–47. Springer International Publishing, Cham.

A rapid and simple method for assessing and representing genome sequence relatednessM Briand, M Bouzid, G Hunault, M Legeay, M Fischer-Le Saux, M Barret<p>Coherent genomic groups are frequently used as a proxy for bacterial species delineation through computation of overall genome relatedness indices (OGRI). Average nucleotide identity (ANI) is a widely employed method for estimating relatedness ...Bioinformatics, MetagenomicsB. Jesse Shapiro Gavin Douglas2019-11-07 16:37:56 View
24 Feb 2023
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MacSyFinder v2: Improved modelling and search engine to identify molecular systems in genomes

A unique and customizable approach for functionally annotating prokaryotic genomes

Recommended by based on reviews by Kwee Boon Brandon Seah and Max Emil Schön

Macromolecular System Finder (MacSyFinder) v2 (Néron et al., 2023) is a newly updated approach for performing functional annotation of prokaryotic genomes (Abby et al., 2014). This tool parses an input file of protein sequences from a single genome (either ordered by genome location or unordered) and identifies the presence of specific cellular functions (referred to as “systems”). These systems are called based on two criteria: (1) that the "quorum" of a minimum set of core proteins involved is reached the “quorum” of a minimum set of core proteins being involved that are present, and (2) that the genes encoding these proteins are in the expected genomic organization (e.g., within the same order in an operon), when ordered data is provided. I believe the MacSyFinder approach represents an improvement over more commonly used methods exactly because it can incorporate such information on genomic organization, and also because it is more customizable.

Before properly appreciating these points, it is worth noting the norms and key challenges surrounding high-throughput functional annotation of prokaryotic genomes. Genome sequences are being added to online repositories at increasing rates, which has led to an enormous amount of bacterial genome diversity available to investigate (Altermann et al., 2022). A key aspect of understanding this diversity is the functional annotation step, which enables genes to be grouped into more biologically interpretable categories. For instance, gene calls can be mapped against existing Clusters of Orthologous Genes, which are themselves grouped into general categories such as ‘Transcription’ and ‘Lipid metabolism’ (Galperin et al., 2021).

This approach is valuable but is primarily used for global summaries of functional annotations within a genome: for example, it could be useful to know that a genome is particularly enriched for genes involved in lipid metabolism. However, knowing that a particular gene is involved in the general process of lipid metabolism is less likely to be actionable. In other words, the desired specificity of a gene’s functional annotation will depend on the exact question being investigated. There is no shortage of functional ontologies in genomics that can be applied for this purpose (Douglas and Langille, 2021), and researchers are often overwhelmed by the choice of which functional ontology to use. In this context, giving researchers the ability to precisely specify the gene families and operon structures they are interested in identifying across genomes provides useful control over what precise functions they are profiling. Of course, most researchers will lack the information and/or expertise to fully take advantage of MacSyFinder’s customizable features, but having this option for specialized purposes is valuable.

The other MacSyFinder feature that I find especially noteworthy is that it can incorporate genomic organization (e.g., of genes ordered in operons) when calling systems. This is a rare feature among commonly used tools for functional annotation and likely results in much higher specificity. As the authors note, this capability makes the co-occurrence of paralogs, and other divergent genes that share sequence similarity, to contribute less noise (i.e., they result in fewer false positive calls).

It is important to emphasize that these features are not new additions in MacSyFinder v2, but there are many other valuable changes. Most practically, this release is written in Python 3, rather than the obsolete Python 2.7, and was made more computationally efficient, which will enable MacSyFinder to be more widely used and more easily maintained moving forward. In addition, the search algorithm for analyzing individual proteins was fundamentally updated as well. The authors show that their improvements to the search algorithm result in an 8% and 20% increase in the number of identified calls for single and multi-locus secretion systems, respectively. Taken together, MacSyFinder v2 represents both practical and scientific improvements over the previous version, which will be of great value to the field. 


Abby SS, Néron B, Ménager H, Touchon M, Rocha EPC (2014) MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems. PLOS ONE, 9, e110726.

Altermann E, Tegetmeyer HE, Chanyi RM (2022) The evolution of bacterial genome assemblies - where do we need to go next? Microbiome Research Reports, 1, 15.

Douglas GM, Langille MGI (2021) A primer and discussion on DNA-based microbiome data and related bioinformatics analyses. Peer Community Journal, 1.

Galperin MY, Wolf YI, Makarova KS, Vera Alvarez R, Landsman D, Koonin EV (2021) COG database update: focus on microbial diversity, model organisms, and widespread pathogens. Nucleic Acids Research, 49, D274–D281.

Néron B, Denise R, Coluzzi C, Touchon M, Rocha EPC, Abby SS (2023) MacSyFinder v2: Improved modelling and search engine to identify molecular systems in genomes. bioRxiv, 2022.09.02.506364, ver. 2 peer-reviewed and recommended by Peer Community in Genomics.

MacSyFinder v2: Improved modelling and search engine to identify molecular systems in genomesBertrand Néron, Rémi Denise, Charles Coluzzi, Marie Touchon, Eduardo P. C. Rocha, Sophie S. Abby<p>Complex cellular functions are usually encoded by a set of genes in one or a few organized genetic loci in microbial genomes. Macromolecular System Finder (MacSyFinder) is a program that uses these properties to model and then annotate cellular...Bacteria and archaea, Bioinformatics, Functional genomicsGavin Douglas Kwee Boon Brandon Seah, Max Emil Schön2022-09-09 10:30:31 View
23 Aug 2022
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A novel lineage of the Capra genus discovered in the Taurus Mountains of Turkey using ancient genomics

Goat ancient DNA analysis unveils a new lineage that may have hybridized with domestic goats

Recommended by based on reviews by Torsten Günther and 1 anonymous reviewer

The genomic analysis of ancient remains has revolutionized the study of the past over the last decade. On top of the discoveries related to human evolution, plant and animal archaeogenomics has been used to gain new insights into the domestication process and the dispersal of domestic forms.

In this study, Daly and colleagues analyse the genomic data from seven goat specimens from the Epipalaeolithic recovered from the Direkli Cave in the Taurus Mountains in southern Turkey. They also generate new genomic data from Capra lineages across the phylogeny, contributing to the availability of genomic resources for this genus. Analysis of the ancient remains is compared to modern genomic variability and sheds light on the complexity of the Tur wild Capra lineages and their relationship with domestic goats and their wild ancestors.

Authors find that during the Late Pleistocene in the Taurus Mountains wild goats from the Tur lineage, today restricted to the Caucasus region, were not rare and cohabited with Bezoar, the wild goats that are the ancestors of domestic goats. They identify the Direkli Cave specimens as a lineage separate from the 
West and East Caucasus Tur modern lineages. Also, analysis of the genomic data and mitochondrial haplotypes reveals hybridization between the Tur and the Bezoar wild lineages. Interestingly, authors also find an uneven amount of Tur ancestry among Neolithic domestic goats, with European domestic goats showing evidence of this ancient Tur ancestry, whereas Neolithic Iranian domestic goats do not, a pattern that is also observed in some modern European domestic goats.

A modified D statistic, Dex, is developed to examine the contribution of the ancient Tur lineage in domestic goats through time and space. Dex measures the relative degree of allele sharing, derived specifically in a selected genome or group of genomes, and may have some utility in genera with complex admixture histories or admixture from ghost lineages. Results confirm that Neolithic European goat had an excess of allele sharing with this ancient Tur lineage, something that is absent in contemporary goats eastwards or in modern goats.

Interspecific gene flow is not uncommon among mammals, but the case of Capra has the additional motivation of understanding the origins of the domestic species. This work uncovers an ancient Tur lineage that is different from the modern ones and is additionally found in another geographic area. Furthermore, evidence shows that this ancient lineage exhibits substantial amounts of allele sharing with the wild ancestor of the domestic goat, but also with the Neolithic Eurasian domestic goats, highlighting the complexity of the domestication process.

This work has also important implications in understanding the effect of over-hunting and habitat disruption during the Anthropocene on the evolution of the Capra genus. The availability of more ancient specimens and better coverage of the modern genomic variability can help quantifying the lineages that went lost and identify the causes of their extinction.

This work is limited by the current availability of whole genomes from modern Capra specimens, but pieces of evidence as well that an effort is needed to obtain more genomic data from ancient goats from different geographic ranges to determine to what extent these lineages contributed to goat domestication.


Daly KG, Arbuckle BS, Rossi C, Mattiangeli V, Lawlor PA, Mashkour M, Sauer E, Lesur J, Atici L, Cevdet CM and Bradley DG (2022) A novel lineage of the Capra genus discovered in the Taurus Mountains of Turkey using ancient genomics. bioRxiv, 2022.04.08.487619, ver. 5 peer-reviewed and recommended by Peer Community in Genomics.

A novel lineage of the Capra genus discovered in the Taurus Mountains of Turkey using ancient genomicsKevin G. Daly, Benjamin S. Arbuckle, Conor Rossi, Valeria Mattiangeli, Phoebe A. Lawlor, Marjan Mashkour, Eberhard Sauer, Joséphine Lesur, Levent Atici, Cevdet Merih Erek, Daniel G. Bradley<p>Direkli Cave, located in the Taurus Mountains of southern Turkey, was occupied by Late Epipaleolithic hunters-gatherers for the seasonal hunting and processing of game including large numbers of wild goats. We report genomic data from new and p...Evolutionary genomics, Population genomics, VertebratesLaura Botigué2022-04-15 12:05:47 View
09 Oct 2020
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An evaluation of pool-sequencing transcriptome-based exon capture for population genomics in non-model species

Assessing a novel sequencing-based approach for population genomics in non-model species

Recommended by and based on reviews by Valentin Wucher and 1 anonymous reviewer

Developing new sequencing and bioinformatic strategies for non-model species is of great interest in many applications, such as phylogenetic studies of diverse related species, but also for studies in population genomics, where a relatively large number of individuals is necessary. Different approaches have been developed and used in these last two decades, such as RAD-Seq (e.g., Miller et al. 2007), exome sequencing (e.g., Teer and Mullikin 2010) and other genome reduced representation methods that avoid the use of a good reference and well annotated genome (reviewed at Davey et al. 2011). However, population genomics studies require the analysis of numerous individuals, which makes the studies still expensive. Pooling samples was thought as an inexpensive strategy to obtain estimates of variability and other related to the frequency spectrum, thus allowing the study of variability at population level (e.g., Van Tassell et al. 2008), although the major drawback was the loss of information related to the linkage of the variants. In addition, population analysis using all these sequencing strategies require statistical and empirical validations that are not always fully performed. A number of studies aiming to obtain unbiased estimates of variability using reduced representation libraries and/or with pooled data have been performed (e.g., Futschik and Schlötterer 2010, Gautier et al. 2013, Ferretti et al. 2013, Lynch et al. 2014), as well as validation of new sequencing methods for population genetic analyses (e.g., Gautier et al. 2013, Nevado et al. 2014). Nevertheless, empirical validation using both pooled and individual experimental approaches combined with different bioinformatic methods has not been always performed.
Here, Deleury et al. (2020) proposed an efficient and elegant way of quantifying the single-nucleotide polymorphisms (SNPs) of exon-derived sequences in a non-model species (i.e. for which no reference genome sequence is available) at the population level scale. They also designed a new procedure to capture exon-derived sequences based on a reference transcriptome. In addition, they were able to make predictions of intron-exon boundaries for de novo transcripts based on the decay of read depth at the ends of the coding regions.
Based on theoretical predictions (Gautier et al. 2013), Deleury et al. (2020) designed a procedure to test the accuracy of variant allele frequencies (AFs) with pooled samples, in a reduced genome-sequence library made with transcriptome regions, and additionally testing the effects of new bioinformatic methods in contrast to standardized methods. They applied their strategy on the non-model species Asian ladybird (Harmonia axyridis), for which a draft genome is available, thereby allowing them to benchmark their method with regard to a traditional mapping-based approach. Based on species-specific de novo transcriptomes, they designed capture probes which are then used to call SNPx and then compared the resulting SNP AFs at the individual (multiplexed) versus population (pooled) levels. Interestingly, they showed that SNP AFs in the pool sequencing strategy nicely correlate with the individual ones but obviously in a cost-effective way. Studies of population genomics for non-model species have usually limited budgets. The number of individuals required for population genomics analysis multiply the costs of the project, making pooling samples an interesting option. Furthermore, the use of pool sequencing is not always a choice, as many organisms are too small and/or individuals are too sticked each other to be individually sequenced (e.g., Choquet et al. 2019, Kurland et al. 2019). In addition, the study of a reduced section of the genome is cheaper and often sufficient for a number of population genetic questions, such as the understanding of general demographic events, or the estimation of the effects of positive and/or negative selection at functional coding regions. Studies on population genomics of non-model species have many applications in related fields, such as conservation genetics, control of invasive species, etc. The work of Deleury et al. (2020) is an elegant contribution to the assessment and validation of new methodologies used for the analysis of genome variations at the intra-population variability level, highlighting straight bioinformatic and reliable sequencing methods for population genomics studies.


[1] Choquet et al. (2019). Towards population genomics in non-model species with large genomes: a case study of the marine zooplankton Calanus finmarchicus. Royal Society open science, 6(2), 180608. doi:
[2] Davey, J. W., Hohenlohe, P. A., Etter, P. D., Boone, J. Q., Catchen, J. M. and Blaxter, M. L. (2011). Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nature Reviews Genetics, 12(7), 499-510. doi:
[3] Deleury, E., Guillemaud, T., Blin, A. and Lombaert, E. (2020) An evaluation of pool-sequencing transcriptome-based exon capture for population genomics in non-model species. bioRxiv, 10.1101/583534, ver. 7 peer-reviewed and recommended by PCI Genomics.
[4] Ferretti, L., Ramos‐Onsins, S. E. and Pérez‐Enciso, M. (2013). Population genomics from pool sequencing. Molecular ecology, 22(22), 5561-5576. doi:
[5] Futschik, A. and Schlötterer, C. (2010). Massively parallel sequencing of pooled DNA samples—the next generation of molecular markers. Genetics, 186 (1), 207-218. doi:
[6] Gautier et al. (2013). Estimation of population allele frequencies from next‐generation sequencing data: pool‐versus individual‐based genotyping. Molecular Ecology, 22(14), 3766-3779. doi:
[7] Kurland et al. (2019). Exploring a Pool‐seq‐only approach for gaining population genomic insights in nonmodel species. Ecology and evolution, 9(19), 11448-11463. doi:
[8] Lynch, M., Bost, D., Wilson, S., Maruki, T. and Harrison, S. (2014). Population-genetic inference from pooled-sequencing data. Genome biology and evolution, 6(5), 1210-1218. doi:
[9] Miller, M. R., Dunham, J. P., Amores, A., Cresko, W. A. and Johnson, E. A. (2007). Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers. Genome research, 17(2), 240-248. doi:
[10] Nevado, B., Ramos‐Onsins, S. E. and Perez‐Enciso, M. (2014). Resequencing studies of nonmodel organisms using closely related reference genomes: optimal experimental designs and bioinformatics approaches for population genomics. Molecular ecology, 23(7), 1764-1779. doi:
[11] Teer, J. K. and Mullikin, J. C. (2010). Exome sequencing: the sweet spot before whole genomes. Human molecular genetics, 19(R2), R145-R151. doi:
[12] Van Tassell et al. (2008). SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nature methods, 5(3), 247-252. doi:

An evaluation of pool-sequencing transcriptome-based exon capture for population genomics in non-model speciesEmeline Deleury, Thomas Guillemaud, Aurélie Blin & Eric Lombaert<p>Exon capture coupled to high-throughput sequencing constitutes a cost-effective technical solution for addressing specific questions in evolutionary biology by focusing on expressed regions of the genome preferentially targeted by selection. Tr...Bioinformatics, Population genomicsThomas Derrien2020-02-26 09:21:11 View
11 Mar 2021
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Gut microbial ecology of Xenopus tadpoles across life stages

A comprehensive look at Xenopus gut microbiota: effects of feed, developmental stages and parental transmission

Recommended by based on reviews by Vanessa Marcelino and 1 anonymous reviewer

It is well established that the gut microbiota play an important role in the overall health of their hosts (Jandhyala et al. 2015). To date, there are still a limited number of studies on the complex microbial communites inhabiting vertebrate digestive systems, especially the ones that also explored the functional diversity of the microbial community (Bletz et al. 2016).

This preprint by Scalvenzi et al. (2021) reports a comprehensive study on the phylogenetic and metabolic profiles of the Xenopus gut microbiota. The author describes significant changes in the gut microbiome communities at different developmental stages and demonstrates different microbial community composition across organs. In addition, the study also investigates the impact of diet on the Xenopus tadpole gut microbiome communities as well as how the bacterial communities are transmitted from parents to the next generation.

This is one of the first studies that addresses the interactions between gut bacteria and tadpoles during the development. The authors observe the dynamics of gut microbiome communities during tadpole growth and metamorphosis. They also explore host-gut microbial community metabolic interactions and demostrate the capacity of the microbiome to complement the metabolic pathways of the Xenopus genome. Although this study is limited by the use of Xenopus tadpoles in a laboratory, which are probably different from those in nature, I believe it still provides important and valuable information for the research community working on vertebrate’s microbiota and their interaction with the host. 


Bletz et al. (2016). Amphibian gut microbiota shifts differentially in community structure but converges on habitat-specific predicted functions. Nature Communications, 7(1), 1-12. doi:

Jandhyala, S. M., Talukdar, R., Subramanyam, C., Vuyyuru, H., Sasikala, M., & Reddy, D. N. (2015). Role of the normal gut microbiota. World journal of gastroenterology: WJG, 21(29), 8787. doi:

Scalvenzi, T., Clavereau, I., Bourge, M. & Pollet, N. (2021) Gut microbial ecology of Xenopus tadpoles across life stages. bioRxiv, 2020.05.25.110734, ver. 4 peer-reviewed and recommended by Peer community in Geonmics.

Gut microbial ecology of Xenopus tadpoles across life stagesThibault Scalvenzi, Isabelle Clavereau, Mickael Bourge, Nicolas Pollet<p><strong>Background</strong> The microorganism world living in amphibians is still largely under-represented and under-studied in the literature. Among anuran amphibians, African clawed frogs of the Xenopus genus stand as well-characterized mode...Evolutionary genomics, Metagenomics, VertebratesWirulda Pootakham2020-05-25 14:01:19 View
08 Nov 2022
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Somatic mutation detection: a critical evaluation through simulations and reanalyses in oaks

How to best call the somatic mosaic tree?

Recommended by based on reviews by 2 anonymous reviewers

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.


Schoen DJ, Schultz ST (2019) Somatic Mutation and Evolution in Plants. Annual Review of Ecology, Evolution, and Systematics, 50, 49–73.

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.

Somatic mutation detection: a critical evaluation through simulations and reanalyses in oaksSylvain Schmitt, Thibault Leroy, Myriam Heuertz, Niklas Tysklind<p style="text-align: justify;">1. Mutation, the source of genetic diversity, is the raw material of evolution; however, the mutation process remains understudied, especially in plants. Using both a simulation and reanalysis framework, we set out ...Bioinformatics, PlantsNicolas BierneAnonymous, Anonymous2022-04-28 13:24:19 View
27 Apr 2021
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Uncovering transposable element variants and their potential adaptive impact in urban populations of the malaria vector Anopheles coluzzii

Anopheles coluzzii, a new system to study how transposable elements may foster adaptation to urban environments

Recommended by based on reviews by Yann Bourgeois and 1 anonymous reviewer

Transposable elements (TEs) are mobile DNA sequences that can increase their copy number and move from one location to another within the genome [1]. Because of their transposition dynamics, TEs constitute a significant fraction of eukaryotic genomes. TEs are also known to play an important functional role and a wealth of studies has now reported how TEs may influence single host traits [e.g. 2–4]. Given that TEs are more likely than classical point mutations to cause extreme changes in gene expression and phenotypes, they might therefore be especially prone to produce the raw diversity necessary for individuals to respond to challenging environments [5,6] such as the ones found in urban area.  
In their study [7], Vargas et al. establish the foundation to investigate how TEs may help Anopheles coluzzii -  the primary vectors of human malaria in sub-Saharan Africa - adapt to urban environments. To cover natural breeding sites in major Central Africa cities, they made use of the previously available An. coluzzii genome from Yaoundé (Cameroon) and sequenced with long-read technology six additional ones originating from Douala (Cameroon) and Libreville (Gabon). The de novo annotation of TEs in these genomes revealed 64 new anopheline TE families and allowed to identify seven active families. As a first step towards characterizing the potential role of TEs in the adaptation of An. coluzzii to urban environments, they further analyzed the distribution of TEs across the seven genomes. By doing so, they identified a significant number of polymorphic or fixed TE insertions located in the vicinity of genes involved in insecticide resistance and immune response genes.  
The availability of seven An. coluzzii genomes allowed the authors to explore how TE diversity may affect genes functionally relevant for the adaptation to urban environments and provide ground for further functional validation studies. More and more studies have demonstrated the impact of TEs on adaptation and as such, the work of Vargas et al. contributes to fostering our understanding of the link between TEs and gain of function in a species facing strong anthropogenic pressures.  
[1] Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, SanMiguel P, Schulman AH (2007) A unified classification system for eukaryotic transposable elements. Nature Reviews Genetics, 8, 973–982.    
[2] van’t Hof AE, Campagne P, Rigden DJ, Yung CJ, Lingley J, Quail MA, Hall N, Darby AC, Saccheri IJ (2016) The industrial melanism mutation in British peppered moths is a transposable element. Nature, 534, 102–105.    
[3] González J, Karasov TL, Messer PW, Petrov DA (2010) Genome-wide patterns of adaptation to temperate environments associated with transposable elements in Drosophila. PLOS Genetics, 6, e1000905.  
[4] Lisch D (2013) How important are transposons for plant evolution? Nature Reviews Genetics, 14, 49–61.    
[5] Bonchev G, Parisod C (2013) Transposable elements and microevolutionary changes in natural populations. Molecular Ecology Resources, 13, 765–775.  
[6] Casacuberta E, González J (2013) The impact of transposable elements in environmental adaptation. Molecular Ecology, 22, 1503–1517.    
[7] Vargas-Chavez C, Pendy NML, Nsango SE, Aguilera L, Ayala D, González J (2021). Uncovering transposable element variants and their potential adaptive impact in urban populations of the malaria vector Anopheles coluzzii. bioRxiv, 2020.11.22.393231, ver. 3 peer-reviewed and recommended by Peer community in Genomics.  


Uncovering transposable element variants and their potential adaptive impact in urban populations of the malaria vector Anopheles coluzziiCarlos Vargas-Chavez, Neil Michel Longo Pendy, Sandrine E. Nsango, Laura Aguilera, Diego Ayala, and Josefa González<p style="text-align: justify;">Background</p> <p style="text-align: justify;">Anopheles coluzzii is one of the primary vectors of human malaria in sub-Saharan Africa. Recently, it has colonized the main cities of Central Africa threatening vecto...Evolutionary genomicsAnne Roulin2020-12-02 14:58:47 View
23 Mar 2022
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Chromosomal rearrangements with stable repertoires of genes and transposable elements in an invasive forest-pathogenic fungus

Comparative genomics in the chestnut blight fungus Cryphonectria parasitica reveals large chromosomal rearrangements and a stable genome organization

Recommended by based on reviews by Benjamin Schwessinger and 1 anonymous reviewer

About twenty-five years after the sequencing of the first fungal genome and a dozen years after the first plant pathogenic fungi genomes were sequenced, unprecedented international efforts have led to an impressive collection of genomes available for the community of mycologists in international databases (Goffeau et al. 1996, Dean et al. 2005; Spatafora et al. 2017). For instance, to date, the Joint Genome Institute Mycocosm database has collected more than 2,100 fungal genomes over the fungal tree of life ( Such resources are paving the way for comparative genomics, population genomics and phylogenomics to address a large panel of questions regarding the biology and the ecology of fungal species. Early on, population genomics applied to pathogenic fungi revealed a great diversity of genome content and organization and a wide variety of variants and rearrangements (Raffaele and Kamoun 2012, Hartmann 2022). Such plasticity raises questions about how to choose a representative genome to serve as an ideal reference to address pertinent biological questions.

Cryphonectria parasitica is a fungal pathogen that is infamous for the devastation of chestnut forests in North America after its accidental introduction more than a century ago (Anagnostakis 1987). Since then, it has been a quarantine species under surveillance in various parts of the world. As for other fungi causing diseases on forest trees, the study of adaptation to its host in the forest ecosystem and of its reproduction and dissemination modes is more complex than for crop-targeting pathogens. A first reference genome was published in 2020 for the chestnut blight fungus C. parasitica strain EP155 in the frame of an international project with the DOE JGI (Crouch et al. 2020). Another genome was then sequenced from the French isolate YVO003, which showed a few differences in the assembly suggesting possible rearrangements (Demené et al. 2019). Here the sequencing of a third isolate ESM015 from the native area of C. parasitica in Japan allows to draw broader comparative analysis and particularly to compare between native and introduced isolates (Demené et al. 2022).

Demené and collaborators report on a new genome sequence using up-to-date long-read sequencing technologies and they provide an improved genome assembly. Comparison with previously published C. parasitica genomes did not reveal dramatic changes in the overall chromosomal landscapes, but large rearrangements could be spotted. Despite these rearrangements, the genome content and organization – i.e. genes and repeats – remain stable, with a limited number of genes gains and losses. As in any fungal plant pathogen genome, the repertoire of candidate effectors predicted among secreted proteins was more particularly scrutinized. Such effector genes have previously been reported in other pathogens in repeat-enriched plastic genomic regions with accelerated evolutionary rates under the pressure of the host immune system (Raffaele and Kamoun 2012). Demené and collaborators established a list of priority candidate effectors in the C. parasitica gene catalog likely involved in the interaction with the host plant which will require more attention in future functional studies. Six major inter-chromosomal translocations were detected and are likely associated with double break strands repairs. The authors speculate on the possible effects that these translocations may have on gene organization and expression regulation leading to dramatic phenotypic changes in relation to introduction and invasion in new continents and the impact regarding sexual reproduction in this fungus (Demené et al. 2022).

I recommend this article not only because it is providing an improved assembly of a reference genome for C. parasitica, but also because it adds diversity in terms of genome references availability, with a third high-quality assembly. Such an effort in the tree pathology community for a pathogen under surveillance is of particular importance for future progress in post-genomic analysis, e.g. in further genomic population studies (Hartmann 2022). 


Anagnostakis SL (1987) Chestnut Blight: The Classical Problem of an Introduced Pathogen. Mycologia, 79, 23–37.

Crouch JA, Dawe A, Aerts A, Barry K, Churchill ACL, Grimwood J, Hillman BI, Milgroom MG, Pangilinan J, Smith M, Salamov A, Schmutz J, Yadav JS, Grigoriev IV, Nuss DL (2020) Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen. Phytopathology®, 110, 1180–1188.

Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu J-R, Pan H, Read ND, Lee Y-H, Carbone I, Brown D, Oh YY, Donofrio N, Jeong JS, Soanes DM, Djonovic S, Kolomiets E, Rehmeyer C, Li W, Harding M, Kim S, Lebrun M-H, Bohnert H, Coughlan S, Butler J, Calvo S, Ma L-J, Nicol R, Purcell S, Nusbaum C, Galagan JE, Birren BW (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature, 434, 980–986.

Demené A., Laurent B., Cros-Arteil S., Boury C. and Dutech C. 2022. Chromosomal rearrangements with stable repertoires of genes and transposable elements in an invasive forest-pathogenic fungus. bioRxiv, 2021.03.09.434572, ver.6 peer-reviewed and recommended by Peer Community in Genomics.

Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 Genes. Science, 274, 546–567.

Hartmann FE (2022) Using structural variants to understand the ecological and evolutionary dynamics of fungal plant pathogens. New Phytologist, 234, 43–49.

Raffaele S, Kamoun S (2012) Genome evolution in filamentous plant pathogens: why bigger can be better. Nature Reviews Microbiology, 10, 417–430.

Spatafora JW, Aime MC, Grigoriev IV, Martin F, Stajich JE, Blackwell M (2017) The Fungal Tree of Life: from Molecular Systematics to Genome-Scale Phylogenies. Microbiology Spectrum, 5, 5.5.03.

Chromosomal rearrangements with stable repertoires of genes and transposable elements in an invasive forest-pathogenic fungusArthur Demene, Benoit Laurent, Sandrine Cros-Arteil, Christophe Boury, Cyril Dutech<p style="text-align: justify;">Chromosomal rearrangements have been largely described among eukaryotes, and may have important consequences on evolution of species. High genome plasticity has been often reported in Fungi, which may explain their ...Evolutionary genomics, FungiSebastien Duplessis2021-03-12 14:18:20 View