Software Choice and Sequencing Coverage Can Impact Plastid Genome Assembly–A Case Study in the Narrow Endemic Calligonum bakuense

Most plastid genome sequences are assembled from short-read whole-genome sequencing data, yet the impact that sequencing coverage and the choice of assembly software can have on the accuracy of the resulting assemblies is poorly understood. In this study, we test the impact of both factors on plastid genome assembly in the threatened and rare endemic shrub Calligonum bakuense. We aim to characterize the differences across plastid genome assemblies generated by different assembly software tools and levels of sequencing coverage and to determine if these differences are large enough to affect the phylogenetic position inferred for C. bakuense compared to congeners. Four assembly software tools (FastPlast, GetOrganelle, IOGA, and NOVOPlasty) and seven levels of sequencing coverage across the plastid genome (original sequencing depth, 2,000x, 1,000x, 500x, 250x, 100x, and 50x) are compared in our analyses. The resulting assemblies are evaluated with regard to reproducibility, contig number, gene complement, inverted repeat length, and computation time; the impact of sequence differences on phylogenetic reconstruction is assessed. Our results show that software choice can have a considerable impact on the accuracy and reproducibility of plastid genome assembly and that GetOrganelle produces the most consistent assemblies for C. bakuense. Moreover, we demonstrate that a sequencing coverage between 500x and 100x can reduce both the sequence variability across assembly contigs and computation time. When comparing the most reliable plastid genome assemblies of C. bakuense, a sequence difference in only three nucleotide positions is detected, which is less than the difference potentially introduced through software choice

Untangling the hedge: Genetic diversity in clonally and sexually transmitted genomes of European wild roses, Rosa L.

While European wild roses are abundant and widely distributed, their morphological taxonomy is complicated and ambiguous. In particular, the polyploid Rosa section Caninae (dogroses) is characterised by its unusual meiosis, causing simultaneous clonal and sexual transmission of sub-genomes. This hemisexual reproduction, which often co-occurs with vegetative reproduction, defies the standard definition of species boundaries. We analysed seven highly polymorphic microsatellite loci, scored for over 2 600 Rosa samples of differing ploidy, collected across Europe within three independent research projects. Based on their morphology, these samples had been identified as belonging to 21 dogrose and five other native rose species. We quantified the degree of clonality within species and at individual sampling sites. We then compared the genetic structure within our data to current rose morpho-systematics and searched for hemisexually co-inherited sets of alleles at individual loci. We found considerably fewer copies of identical multi-locus genotypes in dogroses than in roses with regular meiosis, with some variation recorded among species. While clonality showed no detectable geographic pattern, some genotypes appeared to be more widespread. Microsatellite data confirmed the current classification of subsections, but they did not support most of the generally accepted dogrose microspecies. Under canina meiosis, we found co-inherited sets of alleles as expected, but could not distinguish between sexually and clonally inherited sub-genomes, with only some of the detected allele combinations being lineage-specific

Effects of partial asexuality on the dynamics of genotype frequencies in dominantly diploid populations

Les systèmes reproducteurs déterminent comment le matériel génétique est transmis d’une génération à la suivante […]. Les espèces qui combinent de la reproduction sexuée et asexuée/clonale sont très répandues [… mais] l’effet de leur système reproducteur sur leur évolution reste énigmatique et discuté.L’objectif de cette thèse est de modéliser la dynamique des fréquences génotypiques d’une population avec une combinaison de reproduction sexuée et/ou clonale dans des cycles de vie principalement diploïdes [. … Un] modèle du type chaine de Markov avec temps et états discrets sert de base mathématique pour décrire [leurs] changements […] au cours du temps.Les résultats montrent que la reproduction partiellement asexuée peut en effet modifier la dynamique de la diversité génomique par rapport à une reproduction strictement sexuée ou strictement asexuée. […] L’histoire démographique a un rôle important pour les organismes partiellement clonaux et doit être prise en compte dans toute analyse […].Cette thèse fait des recommandations pour la collecte des données et une hypothèse de base pour l’interprétation des données de génétique/génomique […]. Ces résultats ont des retombées dans plusieurs domaines, allant de la recherche fondamentale […] à des applications en agriculture […], pêche […] et protection de la nature […].Reproductive systems determine how genetic material is passed from one generation to the next, making them an important factor for evolution. Organisms that combine sexual and asexual/clonal reproduction are very widespread [… yet] the effects of their reproductive system on their evolution are still controversial and poorly understood.The aim of this thesis was to model the dynamics of genotype frequencies under combined sexual/clonal reproduction in dominantly diploid life cycles [. … A] state and time discrete Markov chain model served as the mathematical basis to describe [their] changes […] through time.The results demonstrate that partial clonality may indeed change the dynamics of genomic diversity compared to either exclusively sexual or exclusively clonal populations. […] Time has a crucial role in partially clonal populations and needs to be taken into account in any analysis of their genomic diversity.This thesis provides recommendations for data collection and a null hypothesis for the interpretation of population genetic/genomic data […]. Moreover, it includes new methods for the analysis of genotype-based population genetic Markov chain models. These results have a high potential relevance in several areas, ranging from basic research […] to applications in agriculture […], fisheries […] and nature conservation […]

Effets de la reproduction partiellement asexuée sur la dynamique des fréquences génotypiques en populations majoritairement diploïdes

Reproductive systems determine how genetic material is passed from one generation to the next, making them an important factor for evolution. Organisms that combine sexual and asexual/clonal reproduction are very widespread [… yet] the effects of their reproductive system on their evolution are still controversial and poorly understood.The aim of this thesis was to model the dynamics of genotype frequencies under combined sexual/clonal reproduction in dominantly diploid life cycles [. … A] state and time discrete Markov chain model served as the mathematical basis to describe [their] changes […] through time.The results demonstrate that partial clonality may indeed change the dynamics of genomic diversity compared to either exclusively sexual or exclusively clonal populations. […] Time has a crucial role in partially clonal populations and needs to be taken into account in any analysis of their genomic diversity.This thesis provides recommendations for data collection and a null hypothesis for the interpretation of population genetic/genomic data […]. Moreover, it includes new methods for the analysis of genotype-based population genetic Markov chain models. These results have a high potential relevance in several areas, ranging from basic research […] to applications in agriculture […], fisheries […] and nature conservation […].Les systèmes reproducteurs déterminent comment le matériel génétique est transmis d’une génération à la suivante […]. Les espèces qui combinent de la reproduction sexuée et asexuée/clonale sont très répandues [… mais] l’effet de leur système reproducteur sur leur évolution reste énigmatique et discuté.L’objectif de cette thèse est de modéliser la dynamique des fréquences génotypiques d’une population avec une combinaison de reproduction sexuée et/ou clonale dans des cycles de vie principalement diploïdes [. … Un] modèle du type chaine de Markov avec temps et états discrets sert de base mathématique pour décrire [leurs] changements […] au cours du temps.Les résultats montrent que la reproduction partiellement asexuée peut en effet modifier la dynamique de la diversité génomique par rapport à une reproduction strictement sexuée ou strictement asexuée. […] L’histoire démographique a un rôle important pour les organismes partiellement clonaux et doit être prise en compte dans toute analyse […].Cette thèse fait des recommandations pour la collecte des données et une hypothèse de base pour l’interprétation des données de génétique/génomique […]. Ces résultats ont des retombées dans plusieurs domaines, allant de la recherche fondamentale […] à des applications en agriculture […], pêche […] et protection de la nature […]

Updated protocols for the detection of Sotatercept and Luspatercept in human serum

We recently published two protocols for the detection of Sotatercept (ACE-011, ACVR2A-Fc) and Luspatercept (ACE-536, ACVR2B-Fc) in human serum. Both methods used covalently immobilized antibodies on agarose beads for immunoprecipitation and SAR-PAGE/Western blotting for detection. Disadvantages were the relatively high amount of antibody required per sample (10 mu g) and the need of a secondary antibody for the final detection. The updated protocols overcome these limitations by antigen-antibody complex formation in solution followed by capture of the complex with anti-antibody-coated magnetic beads. They also omit the secondary antibody incubation step by usage of biotinylated primary antibodies, which can be directly incubated with streptavidin-HRP. Thus, the new protocols are faster, simpler, and cheaper and offer comparable sensitivities

Detection of Sotatercept (ACE-011) in human serum by SAR-PAGE and western single blotting

A method for the detection of Sotatercept (ACE-011, ACVR2A-Fc) in human serum is presented. The method is a modification of a recently published protocol for Luspatercept (ACE-536, ACVR2B-Fc), another erythropoiesis stimulating fusion protein. Out of 27 tested antibodies against either the extracellular domain of ACVR2A or the full-length protein, only 4 antibodies bound strongly enough to Sotatercept for usage with immunoprecipitation followed by SAR-PAGE and Western single blotting. The adapted protocol allows the detection of 0.1ng/mL Sotatercept in just 50L human serum. None of the 3 commercial ACVR2-ELISAs was able to detect Sotatercept and the 2 tested surrogate proteins, even in the g/mL range. As for Luspatercept, only IPG-IEF/2D-PAGE generated discrete isoforms. Due to the long serum half-life, the SAR-PAGE method will be able to detect Sotatercept for several weeks and will be very useful in doping testing

Python code for Markov chain model

Python code used to compute exact stationary probability distribution of genotypic states

Data from: Effects of complex life cycles on genetic diversity: cyclical parthenogenesis

Neutral patterns of population genetic diversity in species with complex life cycles are difficult to anticipate. Cyclical parthenogenesis (CP), in which organisms undergo several rounds of clonal reproduction followed by a sexual event, is one such life cycle. Many species, including crop pests (aphids), human parasites (trematodes) or models used in evolutionary science (Daphnia), are cyclical parthenogens. It is therefore crucial to understand the impact of such a life cycle on neutral genetic diversity. In this paper, we describe distributions of genetic diversity under conditions of CP with various clonal phase lengths. Using a Markov chain model of CP for a single locus and individual-based simulations for two loci, our analysis first demonstrates that strong departures from full sexuality are observed after only a few generations of clonality. The convergence towards predictions made under conditions of full clonality during the clonal phase depends on the balance between mutations and genetic drift. Second, the sexual event of CP usually resets the genetic diversity at a single locus towards predictions made under full sexuality. However, this single recombination event is insufficient to reshuffle gametic phases towards full-sexuality predictions. Finally, for similar levels of clonality, CP and acyclic partial clonality (wherein a fixed proportion of individuals are clonally produced within each generation) differentially affect the distribution of genetic diversity. Overall, this work provides solid predictions of neutral genetic diversity that may serve as a null model in detecting the action of common evolutionary or demographic processes in cyclical parthenogens (for example, selection or bottlenecks)

Combined detection of the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc) in serum by means of immunoaffinity purification, tryptic digestion, and LC-MS/MS

Therapeutic proteins are a continuously growing class of pharmaceuticals and comprise several drug candidates with potential performance-enhancing properties. In particular, activin receptor competitors, such as the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc), have the potential for being misused as doping agents in sports as they were found to inhibit negative regulators of late-stage erythropoiesis. Within this study, ammonium sulfate precipitation, immunoaffinity purification, tryptic digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to develop an assay for the combined detection of Sotatercept and Luspatercept in doping control serum samples. The assay was optimized, comprehensively characterized, and found to be fit-for-purpose for application to sports drug testing. It complements existing tests for ActRII-Fc fusion proteins and expands the range of available detection methods for novel protein therapeutics