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GigaScience Nov 2019Producing cost-effective haplotype-resolved personal genomes remains challenging. 10x Linked-Read sequencing, with its high base quality and long-range information, has...
BACKGROUND
Producing cost-effective haplotype-resolved personal genomes remains challenging. 10x Linked-Read sequencing, with its high base quality and long-range information, has been demonstrated to facilitate de novo assembly of human genomes and variant detection. In this study, we investigate in depth how the parameter space of 10x library preparation and sequencing affects assembly quality, on the basis of both simulated and real libraries.
RESULTS
We prepared and sequenced eight 10x libraries with a diverse set of parameters from standard cell lines NA12878 and NA24385 and performed whole-genome assembly on the data. We also developed the simulator LRTK-SIM to follow the workflow of 10x data generation and produce realistic simulated Linked-Read data sets. We found that assembly quality could be improved by increasing the total sequencing coverage (C) and keeping physical coverage of DNA fragments (CF) or read coverage per fragment (CR) within broad ranges. The optimal physical coverage was between 332× and 823× and assembly quality worsened if it increased to >1,000× for a given C. Long DNA fragments could significantly extend phase blocks but decreased contig contiguity. The optimal length-weighted fragment length (W${\mu _{FL}}$) was ∼50-150 kb. When broadly optimal parameters were used for library preparation and sequencing, ∼80% of the genome was assembled in a diploid state.
CONCLUSIONS
The Linked-Read libraries we generated and the parameter space we identified provide theoretical considerations and practical guidelines for personal genome assemblies based on 10x Linked-Read sequencing.
Topics: Diploidy; Gene Library; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 31769805
DOI: 10.1093/gigascience/giz141 -
Molecular Biology and Evolution Aug 2023The masking theory states that genes expressed in a haploid stage will be under more efficient selection. In contrast, selection will be less efficient in genes...
The masking theory states that genes expressed in a haploid stage will be under more efficient selection. In contrast, selection will be less efficient in genes expressed in a diploid stage, where the fitness effects of recessive deleterious or beneficial mutations can be hidden from selection in heterozygous form. This difference can influence several evolutionary processes such as the maintenance of genetic variation, adaptation rate, and genetic load. Masking theory expectations have been confirmed in single-cell haploid and diploid organisms. However, in multicellular organisms, such as plants, the effects of haploid selection are not clear-cut. In plants, the great majority of studies indicating haploid selection have been carried out using male haploid tissues in angiosperms. Hence, evidence in these systems is confounded with the effects of sexual selection and intraspecific competition. Evidence from other plant groups is scarce, and results show no support for the masking theory. Here, we have used a gymnosperm Scots pine megagametophyte, a maternally derived seed haploid tissue, and four diploid tissues to test the strength of purifying selection on a set of genes with tissue-specific expression. By using targeted resequencing data of those genes, we obtained estimates of genetic diversity, the site frequency spectrum of 0-fold and 4-fold sites, and inferred the distribution of fitness effects of new mutations in haploid and diploid tissue-specific genes. Our results show that purifying selection is stronger for tissue-specific genes expressed in the haploid megagametophyte tissue and that this signal of strong selection is not an artifact driven by high expression levels.
Topics: Haploidy; Selection, Genetic; Mutation; Biological Evolution; Diploidy; Plants
PubMed: 37565532
DOI: 10.1093/molbev/msad183 -
STAR Protocols Sep 2020Murine cardiomyocytes undergo proliferation, multinucleation, and polyploidization during the first 3 weeks of postnatal life, resulting in a mixture of diploid and...
Murine cardiomyocytes undergo proliferation, multinucleation, and polyploidization during the first 3 weeks of postnatal life, resulting in a mixture of diploid and tetraploid cardiomyocytes in the heart. Understanding the molecular differences between diploid and tetraploid cardiomyocytes from these processes has been limited due to lack of unique markers and their heterogenous origins. Here, we apply single-nucleus RNA-sequencing to fluorescence-activated cell sorting-selected diploid and tetraploid cardiomyocytes to characterize their heterogeneity and molecular distinctions. For complete details on the use and execution of this protocol, please refer to Cui et al. (2020).
Topics: Animals; Cell Culture Techniques; Cells, Cultured; Diploidy; Gene Expression Profiling; Mice; Myocytes, Cardiac; Single-Cell Analysis; Tetraploidy; Transcriptome
PubMed: 33111095
DOI: 10.1016/j.xpro.2020.100049 -
Cytogenetic and Genome Research 2013The loach (Misgurnus anguillicaudatus) is an excellent animal model to elucidate biological origin and evolutionary significance of genome duplication and unisexual... (Review)
Review
The loach (Misgurnus anguillicaudatus) is an excellent animal model to elucidate biological origin and evolutionary significance of genome duplication and unisexual reproduction because artificially induced and naturally occurring polyploids and parthenogenetic (gynogenetic, androgenetic) animals can be compared. First, we summarize the chromosome manipulation techniques to induce triploids and tetraploids by inhibiting meiotic or mitotic divisions of inseminated eggs, respectively, as well as parthenogenetic animals, obtained after fertilization with genetically inactivated gametes. Then, we review the knowledge on natural polyploid and unisexual lineages found in Misgurnus loaches. A natural diploid-tetraploid complex occurs in wild populations in central China, and these diploid and tetraploid loaches reproduce bisexually. Chinese tetraploids are considered autotetraploid, which may have arisen by doubling of the entire genome of an ancestral diploid, based on cytogenetic results from FISH (fluorescence in situ hybridization) karyotypes and meiotic configurations. In contrast, gynogenetically reproducing clonal diploid lineages have been discovered in a few wild populations in Japan, although most wild-type individuals are bisexually reproducing diploids. Such clonal diploid loaches sometimes produce triploid progeny by accidental incorporation of a sperm nucleus into an unreduced diploid egg, and the resulting triploid generates haploid eggs by meiotic hybridogenesis. Unreduced diploid gametes of clonal loaches are generated by a cytological mechanism, premeiotic endomitosis, which likely occurs in the early (gonium stage) germ cells. Initiation of gynogenetic development is related to a failure of decondensation of the male (sperm) pronucleus in unreduced diploid eggs of a clonal loach. Clonal lineages may have arisen from a past hybrid event between genetically divergent groups, but their exact origins are unknown at present. See also the sister article focusing on plants by Hegarty et al. in this themed issue.
Topics: Animals; Cell Nucleus; Chromosome Pairing; Crosses, Genetic; Cypriniformes; Diploidy; Genetic Variation; Hybridization, Genetic; Meiosis; Polyploidy; Reproduction; Sex Chromosomes
PubMed: 23899809
DOI: 10.1159/000353301 -
Canadian Medical Association Journal May 1979
Topics: Animals; Cells, Cultured; Cricetinae; Diploidy; Dogs; Ducks; Embryo, Mammalian; Embryo, Nonmammalian; Humans; Immunization, Secondary; Kidney; Prospective Studies; Rabies; Rabies Vaccines; Vaccination
PubMed: 445294
DOI: No ID Found -
Analytical Chemistry Oct 2017To advance an understanding of cellular regulation and function it is crucial to identify molecular contents in cellular organelles, which accommodate specific...
To advance an understanding of cellular regulation and function it is crucial to identify molecular contents in cellular organelles, which accommodate specific biochemical processes. Toward achievement of this goal, we applied micro-Raman-Biomolecular Component Analysis assay for molecular profiling of major organelles in live cells. We used this assay for comparative analysis of proteins 3D conformation and quantification of proteins, RNA, and lipids concentrations in nucleoli, endoplasmic reticulum, and mitochondria of WI 38 diploid lung fibroblasts and HeLa cancer cells. Obtained data show substantial differences in the concentrations and conformations of proteins in the studied organelles. Moreover, differences in the intraorganellar concentrations of RNA and lipids between these cell lines were found. We report the biological significance of obtained macromolecular profiles and advocate for micro-Raman BCA assay as a valuable proteomics tool.
Topics: Diploidy; Endoplasmic Reticulum; Fibroblasts; HeLa Cells; Humans; Lipids; Mitochondria; Proteins; RNA; Spectrum Analysis, Raman
PubMed: 28910082
DOI: 10.1021/acs.analchem.7b02822 -
American Journal of Botany Jan 2023Strong postzygotic reproductive isolating barriers are usually expected to limit the extent of natural hybridization between species with contrasting ploidy. However,...
PREMISE
Strong postzygotic reproductive isolating barriers are usually expected to limit the extent of natural hybridization between species with contrasting ploidy. However, genomic sequencing has revealed previously overlooked examples of natural cross-ploidy hybridization in some flowering plant genera, suggesting that the phenomenon may be more common than once thought. We investigated potential cross-ploidy hybridization in British eyebrights (Euphrasia, Orobanchaceae), a group from which 13 putative cross-ploidy hybrid combinations have been reported based on morphology.
METHODS
We analyzed a contact zone between diploid Euphrasia rostkoviana and tetraploid E. arctica in Wales. We sequenced part of the internal transcribed spacer (ITS) of nuclear ribosomal DNA and used genotyping by sequencing (GBS) to look for evidence of cross-ploidy hybridization and introgression.
RESULTS
Common variant sites in the ITS region were fixed between diploids and tetraploids, indicating a strong barrier to hybridization. Clustering analyses of 356 single-nucleotide polymorphisms (SNPs) generated using GBS clearly separated samples by ploidy and revealed strong genetic structure (F = 0.44). However, the F distribution across all SNPs was bimodal, indicating potential differential selection on loci between diploids and tetraploids. Demographic inference suggested potential gene flow, limited to around one or fewer migrants per generation.
CONCLUSIONS
Our results suggest that recent cross-ploidy hybridization is rare or absent in a site of secondary contact in Euphrasia. While a strong ploidy barrier prevents hybridization over ecological timescales, such hybrids may form in stable populations over evolutionary timescales, potentially allowing cross-ploidy introgression to take place.
Topics: Diploidy; Tetraploidy; Euphrasia; Ploidies; Hybridization, Genetic
PubMed: 36371731
DOI: 10.1002/ajb2.16100 -
Genome Research Nov 2019Genome assemblies from diploid organisms create mosaic sequences alternating between parental alleles, which can create erroneous gene models and other problems. In...
Genome assemblies from diploid organisms create mosaic sequences alternating between parental alleles, which can create erroneous gene models and other problems. In animals, a popular strategy to generate haploid genome-resolved assemblies has been the sampling of (haploid) gametes, and the advent of single-cell sequencing has further advanced such methods. However, several challenges for the isolation and amplification of DNA from plant gametes have limited such approaches in plants. Here, we combined a new approach for pollen protoplast isolation with a single-cell DNA amplification technique and then used a "barcoding" bioinformatics strategy to incorporate haploid-specific sequence data from 12 pollen cells, ultimately enabling the efficient and accurate phasing of the pear genome into its A and B haploid genomes. Beyond revealing that 8.12% of the genes in the pear reference genome feature mosaic assemblies and enabling a previously impossible analysis of allelic affects in pear gene expression, our new haploid genome assemblies provide high-resolution information about recombination during meiosis in pollen. Considering that outcrossing pear is an angiosperm species featuring very high heterozygosity, our method for rapidly phasing genome assemblies is potentially applicable to several yet-unsequenced outcrossing angiosperm species in nature.
Topics: Computational Biology; DNA, Plant; Diploidy; Genome, Plant; Germ Cells, Plant; Haplotypes; High-Throughput Nucleotide Sequencing; Meiosis; Pollen
PubMed: 31649061
DOI: 10.1101/gr.251033.119 -
PloS One 2017A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a...
A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a 47-year-old Ashkenazi Jewish patient three years after the first surgery removed the primary tumor, both ovaries, and the remaining reproductive organs. OVDM1 was characterized by cell morphology, genotyping, tumorigenic assay, mycoplasma testing, spectral karyotyping (SKY), and molecular profiling of the whole genome by aCGH and gene expression microarray. Targeted sequencing of a panel of cancer-related genes was also performed. Hierarchical clustering of gene expression data clearly confirmed the ovarian origin of the cell line. OVDM1 has a near-diploid karyotype with a low-level aneuploidy, but samples of the original metastatic tumor were grossly aneuploid. A number of single nucleotide variations (SNVs)/mutations were detected in OVDM1 and the metastatic tumor samples. Some of them were cancer-related according to COSMIC and HGMD databases (no founder mutations in BRCA1 and BRCA2 have been found). A large number of focal copy number alterations (FCNAs) were detected, including homozygous deletions (HDs) targeting WWOX and GATA4. Progression of OVDM1 from early to late passages was accompanied by preservation of the near-diploid status, acquisition of only few additional large chromosomal rearrangements and more than 100 new small FCNAs. Most of newly acquired FCNAs seem to be related to localized but massive DNA fragmentation (chromothripsis-like rearrangements). Newly developed near-diploid OVDM1 cell line offers an opportunity to evaluate tumorigenesis pathways/events in a minor clone of metastatic ovarian adenocarcinoma as well as mechanisms of chromothripsis.
Topics: Adult; Aneuploidy; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Diploidy; Disease Progression; Female; Gene Expression Profiling; Genomics; Humans; Mice; Neoplasm Metastasis; Neoplasm Staging; Ovarian Neoplasms
PubMed: 28787462
DOI: 10.1371/journal.pone.0182610 -
Annals of Botany Feb 2023Oil palms showing exceptional vigour and dubbed as 'giant palms' were identified in some progeny during breeding. A panel of phenotypical traits were studied to...
BACKGROUND AND AIMS
Oil palms showing exceptional vigour and dubbed as 'giant palms' were identified in some progeny during breeding. A panel of phenotypical traits were studied to characterize these trees. The hypothesis that gigantism and other anomalies might be linked to polyploidy was investigated.
METHODS
Twenty sib pairs of palms from different crosses, each comprising a giant and a normal oil palm, were studied by flow cytometry with rice 'Nipponbare' as standard reference. In parallel, palms were assessed in the field using 11 phenotypic traits. A principal component analysis (PCA) was conducted to define relationships between these phenotypical traits, and a linear discriminant analysis (LDA) to predict ploidy level and giant classification. Finally, a co-dominant molecular marker study was implemented to highlight the sexual process leading to the formation of 2n gametes.
KEY RESULTS
The first group of oil palms presented an oil palm/rice peak ratio of around 4.8 corresponding to diploid oil palms, whereas the second group presented a ratio of around 7, classifying these plants as triploid. The PCA enabled the classification of the plants in three classes: 21 were normal diploid palms; ten were giant diploid palms; while 11 were giant triploid palms. The LDA revealed three predictors for ploidy classification: phyllotaxy, petiole size and circumference of the plant, but surprisingly not height. The molecular study revealed that triploid palms arose from 2n gametes resulting from the second division restitution of meiosis in parents.
CONCLUSIONS
This study confirms and details the process of sexual polyploidization in oil palm. It also identifies three phenotypical traits to assess the ploidy level of the giant oil palms in the field. In practical terms, our results provide a cheap scientific method to identify polyploid palms in the field.
Topics: Triploidy; Crosses, Genetic; Ploidies; Diploidy; Phenotype; Arecaceae
PubMed: 35299242
DOI: 10.1093/aob/mcac036