Clin.iobio: A Collaborative Diagnostic Workflow to Enable Team-Based Precision Genomics

The primary goal of precision genomics is the identification of causative genetic variants in targeted or whole-genome sequencing data. The ultimate clinical hope is that these findings lead to an efficacious change in treatment for the patient. In current clinical practice, these findings are typically returned by expert analysts as static, text-based reports. Ideally, these reports summarize the quality of the data obtained, integrate known gene–phenotype associations, follow allele segregation and affected status within the sequenced samples, and weigh computational evidence of pathogenicity. These findings are used to prioritize the variant(s) most likely to cause the given patient’s phenotypes. In most diagnostic settings, a team of experts contribute to these reports, including bioinformaticians, clinicians, and genetic counselors, among others. However, these experts often do not have the necessary tools to review genomic findings, test genetic hypotheses, or query specific gene and variant information. Additionally, team members often rely on different tools and methods based on their given expertise, resulting in further difficulties in communicating and discussing genomic findings. Here, we present clin.iobio—a web-based solution to collaborative genomic analysis that enables diagnostic team members to focus on their area of expertise within the diagnostic process, while allowing them to easily review and contribute to all steps of the diagnostic process. Clin.iobio integrates tools from the popular iobio genomic visualization suite into a comprehensive diagnostic workflow, encompassing (1) genomic data quality review, (2) dynamic phenotype-driven gene prioritization, (3) variant prioritization using a comprehensive set of knowledge bases and annotations, (4) and an exportable findings summary. In conclusion, clin.iobio is a comprehensive solution to team-based precision genomics, the findings of which stand to inform genomic considerations in clinical practice.

Allele segregation analysis of F1 hybrids between independent Brassica allohexaploid lineages

In the Brassica genus we find both diploid species (one genome) and allotetraploid species (two different genomes) but no naturally occurring hexaploid species (three different genomes, AABBCC). Although hexaploids can be produced via human intervention, these neo-polyploids have quite unstable genomes and usually suffer from severe genome reshuffling. Whether these genome rearrangements continue in later generations and whether genomic arrangements follow similar, reproducible patterns between different lines is still unknown. We crossed Brassica hexaploids resulting from different species combinations to produce five F1 hybrids, and analyzed the karyotypes of the parents and the F1 hybrids, as well as allele segregation in a resulting test-cross population via molecular karyotyping using SNP array genotyping. Although some genomic regions were found to be more likely to be duplicated, deleted or rearranged, a consensus pattern was not shared between genotypes. Brassica hexaploids had a high tolerance for fixed structural rearrangements, but which rearrangements occur and become fixed over many generations does not seem to show either strong reproducibility or to indicate selection for stability. On average, we observed 10 de novo chromosome rearrangements contributed almost equally from both parents to the F1 hybrids. At the same time, the F1 hybrid meiosis produced on average 8.6 new rearrangements. Hence, the increased heterozygosity in the F1 hybrid did not significantly improve genome stability in our hexaploid hybrids, and might have had the opposite effect. However, hybridization between lineages was readily achieved and may be exploited for future genetics and breeding purposes.

Fifty Generations of Amitosis: Tracing Asymmetric Allele Segregation in Polyploid Cells with Single-Cell DNA Sequencing

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis have been gleaned by assessing the rate of phenotypic assortment. Though powerful, this experimental approach relies on the availability of phenotypic markers. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dualism and a highly polyploid somatic nucleus, we probe the limits of single-cell whole-genome sequencing to study the consequences of amitosis. To this end, we first evaluate the suitability of single-cell sequencing to study the AT-rich genome of P. tetraurelia, focusing on common sources of genome representation bias. We then asked: can alternative rearrangements of a given locus eventually assort after a number of amitotic divisions? To address this question, we track somatic assortment of developmentally acquired Internal Eliminated Sequences (IESs) up to 50 amitotic divisions post self-fertilization. To further strengthen our observations, we contrast empirical estimates of IES retention levels with in silico predictions obtained through mathematical modeling. In agreement with theoretical expectations, our empirical findings are consistent with a mild increase in variation of IES retention levels across successive amitotic divisions of the macronucleus. The modest levels of somatic assortment in P. tetraurelia suggest that IESs retention levels are largely sculpted at the time of macronuclear development, and remain fairly stable during vegetative growth. In forgoing the requirement for phenotypic assortment, our approach can be applied to a wide variety of amitotic species and could facilitate the identification of environmental and genetic factors affecting amitosis.

Characterization of Japanese Plum (Prunus salicina) PsMYB10 Alleles Reveals Structural Variation and Polymorphisms Correlating With Fruit Skin Color

The red to blue hue of plant organs is caused due to anthocyanins, which are water-soluble flavonoid pigments. The accumulation of these pigments is regulated by a complex of R2R3-MYB transcription factors (TFs), basic-helix-loop-helix (bHLH), and WD-repeat (WDR) proteins (MBW complex). In Rosaceae species, R2R3-MYBs, particularly MYB10 genes, are responsible for part of the natural variation in anthocyanin colors. Japanese plum cultivars, which are hybrids of Prunus salicina, have high variability in the color hue and pattern, going from yellow-green to red and purple-blue, probably as a result of the interspecific hybridization origin of the crop. Because of such variability, Japanese plum can be considered as an excellent model to study the color determination in Rosaceae fruit tree species. Here, we cloned and characterized the alleles of the PsMYB10 genes in the linkage group LG3 region where quantitative trait loci (QTLs) for the organ color have been mapped to other Prunus species. Allele segregation in biparental populations as well as in a panel of varieties, combined with the whole-genome sequence of two varieties with contrasting fruit color, allowed the organization of the MYB10 alleles into haplotypes. With the help of this strategy, alleles were assigned to genes and at least three copies of PsMYB10.1 were identified in some varieties. In total, we observed six haplotypes, which were able to characterize 91.36% of the cultivars. In addition, two alleles of PsMYB10.1 were found to be highly associated with anthocyanin and anthocyanin-less skin. Their expression during the fruit development confirms their role in the fruit skin coloration. Here, we provide a highly efficient molecular marker for the early selection of colored or non-colored fruits in Japanese plum breeding programs.

Fifty generations of amitosis: tracing asymmetric allele segregation in polyploid cells with single-cell DNA sequencing

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis are acquired by assessing the rate of phenotypic assortment. The phenotypic diversification of heterozygous clones during successive cell divisions reveals the random segregation of alleles to daughter nuclei. Though powerful, this experimental approach relies on the availability of phenotypic markers. Here, we present an approach that overcomes the requirement for phenotypic assortment. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dimorphism and a highly polyploid somatic nucleus, we use single-cell whole-genome sequencing to track the assortment of developmentally acquired somatic DNA variants. Accounting for genome representation biases, we measure the effect of amitosis on allele segregation across the first ~50 amitotic divisions post self-fertilization and compare our empirical findings with theoretical predictions estimated via mathematical modeling. In line with our simulations, we show that amitosis in P. tetraurelia produces measurable but modest levels of somatic assortment. In forgoing the requirement for phenotypic assortment and employing developmental, environmentally induced somatic variation as molecular markers, our work provides a new powerful approach to investigate the consequences of amitosis in polyploid cells.

Computational Inference Software for Tetrad Assembly from Randomly Arrayed Yeast Colonies

Here, we describe an information-theory-based method and associated software for computationally identifying sister spores derived from the same meiotic tetrad. The method exploits specific DNA sequence features of tetrads that result from meiotic centromere and allele segregation patterns. Because the method uses only the genomic sequence, it alleviates the need for tetrad-specific barcodes or other genetic modifications to the strains. Using this method, strains derived from randomly arrayed spores can be efficiently grouped back into tetrads.

Reactive oxygen species stimulate mitochondrial allele segregation toward homoplasmy in human cells

Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are heteroplasmic. In humans, homoplasmy is restored during early oogenesis and reprogramming of somatic cells, but the mechanism of mt-allele segregation remains unknown. In budding yeast, homoplasmy is restored by head-to-tail concatemer formation in mother cells by reactive oxygen species (ROS)–induced rolling-circle replication and selective transmission of concatemers to daughter cells, but this mechanism is not obvious in higher eukaryotes. Here, using heteroplasmic m.3243A > G primary fibroblast cells derived from MELAS patients treated with hydrogen peroxide (H2O2), we show that an optimal ROS level promotes mt-allele segregation toward wild-type and mutant mtDNA homoplasmy. Enhanced ROS level reduced the amount of intact mtDNA replication templates but increased linear tandem multimers linked by head-to-tail unit-sized mtDNA (mtDNA concatemers). ROS-triggered mt-allele segregation correlated with mtDNA-concatemer production and enabled transmission of multiple identical mt-genome copies as a single unit. Our results support a mechanism by which mt-allele segregation toward mt-homoplasmy is mediated by concatemers.