scholarly journals Heterochromatin-enriched assemblies reveal the sequence and organization of the Drosophila melanogaster Y chromosome

2018 ◽  
Author(s):  
Ching-Ho Chang ◽  
Amanda M. Larracuente
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Family ◽  
Gene Conversion ◽  
Y Chromosome ◽  
Single Molecule ◽  
De Novo ◽  
High Rate ◽  
Genome Wide ◽  
Conversion Rates ◽  
Multiple Copies

ABSTRACTHeterochromatic regions of the genome are repeat-rich and gene poor, and are therefore underrepresented in even in the best genome assemblies. One of the most difficult regions of the genome to assemble are sex-limited chromosomes. The Drosophila melanogaster Y chromosome is entirely heterochromatic, yet has wide-ranging effects on male fertility, fitness, and genome-wide gene expression. The genetic basis of this phenotypic variation is difficult to study, in part because we do not know the detailed organization of the Y chromosome. To study Y chromosome organization in D. melanogaster, we develop an assembly strategy involving the in silico enrichment of heterochromatic long single-molecule reads and use these reads to create targeted de novo assemblies of heterochromatic sequences. We assigned contigs to the Y chromosome using Illumina reads to identify male-specific sequences. Our pipeline extends the D. melanogaster reference genome by 11.9-Mb, closes 43.8% of the gaps, and improves overall contiguity. The addition of 10.6 MB of Y-linked sequence permitted us to study the organization of repeats and genes along the Y chromosome. We detected a high rate of duplication to the pericentric regions of the Y chromosome from other regions in the genome. Most of these duplicated genes exist in multiple copies. We detail the evolutionary history of one sex-linked gene family—crystal-Stellate. While the Y chromosome does not undergo crossing over, we observed high gene conversion rates within and between members of the crystal-Stellate gene family, Su(Ste), and PCKR, compared to genome-wide estimates. Our results suggest that gene conversion and gene duplication play an important role in the evolution of Y-linked genes.

scholarly journals Mapping and phasing of structural variation in patient genomes using nanopore sequencing

2017 ◽  
Author(s):  
Mircea Cretu Stancu ◽  
Markus J. van Roosmalen ◽  
Ivo Renkens ◽  
Marleen Nieboer ◽  
Sjors Middelkamp ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Human Genetic Disease ◽  
Structural Genomic ◽  
Short Read ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Long Read ◽  
Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

scholarly journals Epigenetic conflict on a degenerating Y chromosome increases mutational burden in Drosophila males

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin H.-C. Wei ◽  
Lauren Gibilisco ◽  
Doris Bachtrog
Keyword(s):  
Y Chromosome ◽  
Early Development ◽  
Defense Mechanisms ◽  
De Novo ◽  
Genome Integrity ◽  
Selfish Dna ◽  
Mutational Burden ◽  
Y Chromosomes ◽  
Genome Wide ◽  
Eukaryotic Genomes

Abstract Large portions of eukaryotic genomes consist of transposable elements (TEs), and the establishment of transcription-repressing heterochromatin during early development safeguards genome integrity in Drosophila. Repeat-rich Y chromosomes can act as reservoirs for TEs (‘toxic’ Y effect), and incomplete epigenomic defenses during early development can lead to deleterious TE mobilization. Here, we contrast the dynamics of early TE activation in two Drosophila species with vastly different Y chromosomes of different ages. Zygotic TE expression is elevated in male embryos relative to females in both species, mostly due to expression of Y-linked TEs. Interestingly, male-biased TE expression diminishes across development in D. pseudoobscura, but remains elevated in D. miranda, the species with the younger and larger Y chromosome. The repeat-rich Y of D. miranda still contains many actively transcribed genes, which compromise the formation of silencing heterochromatin. Elevated TE expression results in more de novo insertions of repeats in males compared to females. This lends support to the idea that the ‘toxic’ Y chromosome can create a mutational burden in males when genome-wide defense mechanisms are compromised, and suggests a previously unappreciated epigenetic conflict on evolving Y chromosomes between transcription of essential genes and silencing of selfish DNA.

scholarly journals De novo assembly of a young Drosophila Y chromosome using single-molecule sequencing and chromatin conformation capture

PLoS Biology ◽  
2018 ◽  
Vol 16 (7) ◽  
pp. e2006348 ◽  
Author(s):  
Shivani Mahajan ◽  
Kevin H.-C. Wei ◽  
Matthew J. Nalley ◽  
Lauren Gibilisco ◽  
Doris Bachtrog
Keyword(s):  
Y Chromosome ◽  
Single Molecule ◽  
De Novo Assembly ◽  
De Novo ◽  
Chromatin Conformation ◽  
Single Molecule Sequencing ◽  
Chromatin Conformation Capture

scholarly journals Leveraging distant relatedness to quantify human mutation and gene conversion rates

2015 ◽  
Author(s):  
Pier Francesco Palamara ◽  
Laurent Francioli ◽  
Giulio Genovese ◽  
Peter Wilton ◽  
Alexander Gusev ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Mutation Rate ◽  
Recombination Rates ◽  
Human Genomes ◽  
Coalescent Simulations ◽  
Genome Wide ◽  
Conversion Rates ◽  
Human Mutation ◽  
A Site ◽  
Mutagenic Effects

The rate at which human genomes mutate is a central biological parameter that has many implications for our ability to understand demographic and evolutionary phenomena. We present a method for inferring mutation and gene conversion rates using the number of sequence differences observed in identical-by-descent (IBD) segments together with a reconstructed model of recent population size history. This approach is robust to, and can quantify, the presence of substantial genotyping error, as validated in coalescent simulations. We applied the method to 498 trio-phased Dutch individuals from the Genome of the Netherlands (GoNL) project, sequenced at an average depth of 13×. We infer a point mutation rate of 1.66 ± 0.04 × 10-8per base per generation, and a rate of 1.26 ± 0.06 × 10-9for <20 bp indels. Our estimated average genome-wide mutation rate is higher than most pedigree-based estimates reported thus far, but lower than estimates obtained using substitution rates across primates. By quantifying how estimates vary as a function of allele frequency, we infer the probability that a site is involved in non-crossover gene conversion as 5.99 ± 0.69 × 10-6, consistent with recent reports. We find that recombination does not have observable mutagenic effects after gene conversion is accounted for, and that local gene conversion rates reflect recombination rates. We detect a strong enrichment for recent deleterious variation among mismatching variants found within IBD regions, and observe summary statistics of local IBD sharing to closely match previously proposed metrics of background selection, but find no significant effects of selection on our estimates of mutation rate. We detect no evidence for strong variation of mutation rates in a number of genomic annotations obtained from several recent studies.

scholarly journals Intracellular selection, conversion bias, and the expected substitution rate of organelle genes.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 939-946 ◽  
Author(s):  
J B Walsh
Keyword(s):  
Gene Conversion ◽  
Substitution Rate ◽  
Germ Line ◽  
Genome Replication ◽  
Initial Fixation ◽  
Conversion Rates ◽  
Degradation Rates ◽  
Biased Gene Conversion ◽  
Multiple Copies ◽  
Probability Of Fixation

Abstract A key step in the substitution of a new organelle mutant throughout a population is the generation of germ-line cells homoplasmic for that mutant. Given that each cell typically contains multiple copies of organelles, each of which in turn contains multiple copies of the organelle genome, processes akin to drift and selection in a population are responsible for producing homoplasmic cells. This paper examines the expected substitution rate of new mutants by obtaining the probability that a new mutant is fixed throughout a cell, allowing for arbitrary rates of genome turnover within an organelle and organelle turnover within the cell, as well as (possibly biased) gene conversion and genetic differences in genome and/or organelle replication rates. Analysis is based on a variation of Moran's model for drift in a haploid population. One interesting result is that if the rate of unbiased conversion is sufficiently strong, it creates enough intracellular drift to overcome even strong differences in the replication rates of wild-type and mutant genomes. Thus, organelles with very high conversion rates are more resistant to intracellular selection based on differences in genome replication and/or degradation rates. It is found that the amount of genetic exchange between organelles within the cell greatly influences the probability of fixation. In the absence of exchange, biased gene conversion and/or differences in genome replication rates do not influence the probability of fixation beyond the initial fixation within a single organelle. With exchange, both these processes influence the probability of fixation throughout the entire cell. Generally speaking, exchange between organelles accentuates the effects of directional intracellular forces.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Single-molecule functional anatomy of endogenous HER2-HER3 heterodimers

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Byoungsan Choi ◽  
Minkwon Cha ◽  
Gee Sung Eun ◽  
Dae Hee Lee ◽  
Seul Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Functional Anatomy ◽  
High Rate ◽  
Epidermal Growth Factor Receptors ◽  
Cancer Therapies ◽  
Downstream Signaling ◽  
Multiple Copies ◽  
Epidermal Growth ◽  
The Individual ◽  
Proliferative Signal

Human epidermal growth factor receptors (HERs) are the primary targets of many directed cancer therapies. However, the reason a specific dimer of HERs generates a stronger proliferative signal than other permutations remains unclear. Here, we used single-molecule immunoprecipitation to develop a biochemical assay for endogenously-formed, entire HER2-HER3 heterodimers. We observed unexpected, large conformational fluctuations in juxta-membrane and kinase domains of the HER2-HER3 heterodimer. Nevertheless, the individual HER2-HER3 heterodimers catalyze tyrosine phosphorylation at an unusually high rate, while simultaneously interacting with multiple copies of downstream signaling effectors. Our results suggest that the high catalytic rate and multi-tasking capability make a concerted contribution to the strong signaling potency of the HER2-HER3 heterodimers.

scholarly journals De novo assembly of a young Drosophila Y chromosome using Single-Molecule sequencing and Chromatin Conformation capture

2018 ◽  
Author(s):  
Shivani Mahajan ◽  
Kevin Wei ◽  
Matthew Nalley ◽  
Lauren Giblisco ◽  
Doris Bachtrog
Keyword(s):  
Y Chromosome ◽  
Single Molecule ◽  
Sex Chromosome ◽  
De Novo ◽  
Repetitive Sequences ◽  
Chromatin Interaction ◽  
Bac Clone ◽  
Long Reads ◽  
Y Chromosome Evolution ◽  
Genome Assemblies

While short-read sequencing technology has resulted in a sharp increase in the number of species with genome assemblies, these assemblies are typically highly fragmented. Repeats pose the largest challenge for reference genome assembly, and pericentromeric regions and the repeat-rich Y chromosome are typically ignored from sequencing projects. Here, we assemble the genome of Drosophila miranda using long reads for contig formation, chromatin interaction maps for scaffolding and short reads, optical mapping and BAC clone sequencing for consensus validation. Our assembly recovers entire chromosomes and contains large fractions of repetitive DNA, including ~41.5 Mb of pericentromeric and telomeric regions, and >100Mb of the recently formed highly repetitive neo-Y chromosome. While Y chromosome evolution is typically characterized by global sequence loss and shrinkage, the neo-Y increased in size by almost 3-fold, due to the accumulation of repetitive sequences. Our high-quality assembly allows us to reconstruct the chromosomal events that have led to the unusual sex chromosome karyotype in D. miranda, including the independent de novo formation of a pair of sex chromosomes at two distinct time points, or the reversion of a former Y chromosome to an autosome.

scholarly journals Evolutionary Dynamics of the POTE Gene Family in Human and Nonhuman Primates

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 213
Author(s):  
Flavia Angela Maria Maggiolini ◽  
Ludovica Mercuri ◽  
Francesca Antonacci ◽  
Fabio Anaclerio ◽  
Francesco Maria Calabrese ◽  
...  
Keyword(s):  
Gene Family ◽  
Single Molecule ◽  
Copy Number ◽  
Evolutionary Dynamics ◽  
De Novo ◽  
Specific Gene ◽  
Segmental Duplications ◽  
Chromosomal Distribution ◽  
High Sequence Identity ◽  
Specific Expression

POTE (prostate, ovary, testis, and placenta expressed) genes belong to a primate-specific gene family expressed in prostate, ovary, and testis as well as in several cancers including breast, prostate, and lung cancers. Due to their tumor-specific expression, POTEs are potential oncogenes, therapeutic targets, and biomarkers for these malignancies. This gene family maps within human and primate segmental duplications with a copy number ranging from two to 14 in different species. Due to the high sequence identity among the gene copies, specific efforts are needed to assemble these loci in order to correctly define the organization and evolution of the gene family. Using single-molecule, real-time (SMRT) sequencing, in silico analyses, and molecular cytogenetics, we characterized the structure, copy number, and chromosomal distribution of the POTE genes, as well as their expression in normal and disease tissues, and provided a comparative analysis of the POTE organization and gene structure in primate genomes. We were able, for the first time, to de novo sequence and assemble a POTE tandem duplication in marmoset that is misassembled and collapsed in the reference genome, thus revealing the presence of a second POTE copy. Taken together, our findings provide comprehensive insights into the evolutionary dynamics of the primate-specific POTE gene family, involving gene duplications, deletions, and long interspersed nuclear element (LINE) transpositions to explain the actual repertoire of these genes in human and primate genomes.

scholarly journals A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition

2016 ◽  
Author(s):  
Chris C.-S. Hsiung ◽  
Caroline Bartman ◽  
Peng Huang ◽  
Paul Ginart ◽  
Aaron J. Stonestrom ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
De Novo ◽  
Pol Ii ◽  
Mature Mrna ◽  
Genome Wide ◽  
Transcriptional State ◽  
Active Genes

AbstractDuring mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ~50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry, but do not spike. Of the chromatin-associated features examined, histone H3 lysine 27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.

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