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September 22, 2019

Meiotic drive of female-inherited supernumerary chromosomes in a pathogenic fungus.

Meiosis is a key cellular process of sexual reproduction that includes pairing of homologous sequences. In many species however, meiosis can also involve the segregation of supernumerary chromosomes, which can lack a homolog. How these unpaired chromosomes undergo meiosis is largely unknown. In this study we investigated chromosome segregation during meiosis in the haploid fungus Zymoseptoria tritici that possesses a large complement of supernumerary chromosomes. We used isogenic whole chromosome deletion strains to compare meiotic transmission of chromosomes when paired and unpaired. Unpaired chromosomes inherited from the male parent as well as paired supernumerary chromosomes in general showed Mendelian inheritance. In contrast, unpaired chromosomes inherited from the female parent showed non-Mendelian inheritance but were amplified and transmitted to all meiotic products. We concluded that the supernumerary chromosomes of Z. tritici show a meiotic drive and propose an additional feedback mechanism during meiosis, which initiates amplification of unpaired female-inherited chromosomes.© 2018, Habig et al.

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September 22, 2019

Density-dependent enhanced replication of a densovirus in Wolbachia-infected Aedes cells is associated with production of piRNAs and higher virus-derived siRNAs.

The endosymbiotic bacterium Wolbachia pipientis has been shown to restrict a range of RNA viruses in Drosophila melanogaster and transinfected dengue mosquito, Aedes aegypti. Here, we show that Wolbachia infection enhances replication of Aedes albopictus densovirus (AalDNV-1), a single stranded DNA virus, in Aedes cell lines in a density-dependent manner. Analysis of previously produced small RNAs of Aag2 cells showed that Wolbachia-infected cells produced greater absolute abundance of virus-derived short interfering RNAs compared to uninfected cells. Additionally, we found production of virus-derived PIWI-like RNAs (vpiRNA) produced in response to AalDNV-1 infection. Nuclear fractions of Aag2 cells produced a primary vpiRNA signature U1 bias whereas the typical “ping-pong” signature (U1 – A10) was evident in vpiRNAs from the cytoplasmic fractions. This is the first report of the density-dependent enhancement of DNA viruses by Wolbachia. Further, we report the generation of vpiRNAs in a DNA virus-host interaction for the first time. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

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September 22, 2019

The genomic landscape of molecular responses to natural drought stress in Panicum hallii

Environmental stress is a major driver of ecological community dynamics and agricultural productivity. This is especially true for soil water availability, because drought is the greatest abiotic inhibitor of worldwide crop yields. Here, we test the genetic basis of drought responses in the genetic model for C4perennial grasses, Panicum hallii, through population genomics, field-scale gene-expression (eQTL) analysis, and comparison of two complete genomes. While gene expression networks are dominated by local cis-regulatory elements, we observe three genomic hotspots of unlinked trans-regulatory loci. These regulatory hubs are four times more drought responsive than the genome-wide average. Additionally, cis- and trans-regulatory networks are more likely to have opposing effects than expected under neutral evolution, supporting a strong influence of compensatory evolution and stabilizing selection. These results implicate trans-regulatory evolution as a driver of drought responses and demonstrate the potential for crop improvement in drought-prone regions through modification of gene regulatory networks.

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September 22, 2019

Genomic characterization of a B chromosome in Lake Malawi cichlid fishes.

B chromosomes (Bs) were discovered a century ago, and since then, most studies have focused on describing their distribution and abundance using traditional cytogenetics. Only recently have attempts been made to understand their structure and evolution at the level of DNA sequence. Many questions regarding the origin, structure, function, and evolution of B chromosomes remain unanswered. Here, we identify B chromosome sequences from several species of cichlid fish from Lake Malawi by examining the ratios of DNA sequence coverage in individuals with or without B chromosomes. We examined the efficiency of this method, and compared results using both Illumina and PacBio sequence data. The B chromosome sequences detected in 13 individuals from 7 species were compared to assess the rates of sequence replacement. B-specific sequence common to at least 12 of the 13 datasets were identified as the “Core” B chromosome. The location of B sequence homologs throughout the genome provides further support for theories of B chromosome evolution. Finally, we identified genes and gene fragments located on the B chromosome, some of which may regulate the segregation and maintenance of the B chromosome.

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September 22, 2019

Detection and visualization of complex structural variants from long reads.

With applications in cancer, drug metabolism, and disease etiology, understanding structural variation in the human genome is critical in advancing the thrusts of individualized medicine. However, structural variants (SVs) remain challenging to detect with high sensitivity using short read sequencing technologies. This problem is exacerbated when considering complex SVs comprised of multiple overlapping or nested rearrangements. Longer reads, such as those from Pacific Biosciences platforms, often span multiple breakpoints of such events, and thus provide a way to unravel small-scale complexities in SVs with higher confidence.We present CORGi (COmplex Rearrangement detection with Graph-search), a method for the detection and visualization of complex local genomic rearrangements. This method leverages the ability of long reads to span multiple breakpoints to untangle SVs that appear very complicated with respect to a reference genome. We validated our approach against both simulated long reads, and real data from two long read sequencing technologies. We demonstrate the ability of our method to identify breakpoints inserted in synthetic data with high accuracy, and the ability to detect and plot SVs from NA12878 germline, achieving 88.4% concordance between the two sets of sequence data. The patterns of complexity we find in many NA12878 SVs match known mechanisms associated with DNA replication and structural variant formation, and highlight the ability of our method to automatically label complex SVs with an intuitive combination of adjacent or overlapping reference transformations.CORGi is a method for interrogating genomic regions suspected to contain local rearrangements using long reads. Using pairwise alignments and graph search CORGi produces labels and visualizations for local SVs of arbitrary complexity.

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September 22, 2019

Approaches for surveying cosmic radiation damage in large populations of Arabidopsis thaliana seeds-Antarctic balloons and particle beams.

The Cosmic Ray Exposure Sequencing Science (CRESS) payload system is a proof of concept experiment to assess the genomic impact of space radiation on seeds. CRESS was designed as a secondary payload for the December 2016 high-altitude, high-latitude, and long-duration balloon flight carrying the Boron And Carbon Cosmic Rays in the Upper Stratosphere (BACCUS) experimental hardware. Investigation of the biological effects of Galactic Cosmic Radiation (GCR), particularly those of ions with High-Z and Energy (HZE), is of interest due to the genomic damage this type of radiation inflicts. The biological effects of upper-stratospheric mixed radiation above Antarctica (ANT) were sampled using Arabidopsis thaliana seeds and were compared to those resulting from a controlled simulation of GCR at Brookhaven National Laboratory (BNL) and to laboratory control seed. The payload developed for Antarctica exposure was broadly designed to 1U CubeSat specifications (10cmx10cmx10cm, =1.33kg), maintained 1 atm internal pressure, and carried an internal cargo of four seed trays (about 580,000 seeds) and twelve CR-39 Solid-State Nuclear Track Detectors (SSNTDs). The irradiated seeds were recovered, sterilized and grown on Petri plates for phenotypic screening. BNL and ANT M0 seeds showed significantly reduced germination rates and elevated somatic mutation rates when compared to non-irradiated controls, with the BNL mutation rate also being significantly higher than that of ANT. Genomic DNA from mutants of interest was evaluated with whole-genome sequencing using PacBio SMRT technology. Sequence data revealed the presence of an array of genome structural variants in the genomes of M0 and M1 mutant plants.

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September 22, 2019

Genetics and genomics of an unusual selfish sex ratio distortion in an insect.

Diverse selfish genetic elements have evolved the ability to manipulate reproduction to increase their transmission, and this can result in highly distorted sex ratios [1]. Indeed, one of the major explanations for why sex determination systems are so dynamic is because they are shaped by ongoing coevolutionary arms races between sex-ratio-distorting elements and the rest of the genome [2]. Here, we use genetic crosses and genome analysis to describe an unusual sex ratio distortion with striking consequences on genome organization in a booklouse species, Liposcelis sp. (Insecta: Psocodea), in which two types of females coexist. Distorter females never produce sons but must mate with males (the sons of nondistorting females) to reproduce [3]. Although they are diploid and express the genes inherited from their fathers in somatic tissues, distorter females only ever transmit genes inherited from their mothers. As a result, distorter females have unusual chimeric genomes, with distorter-restricted chromosomes diverging from their nondistorting counterparts and exhibiting features of a giant non-recombining sex chromosome. The distorter-restricted genome has also acquired a gene from the bacterium Wolbachia, a well-known insect reproductive manipulator; we found that this gene has independently colonized the genomes of two other insect species with unusual reproductive systems, suggesting possible roles in sex ratio distortion in this remarkable genetic system. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Relationship between Alzheimer’s disease-associated SNPs within the CLU gene, local DNA methylation and episodic verbal memory in healthy and schizophrenia subjects.

Genetic variation may impact on local DNA methylation patterns. Therefore, information about allele-specific DNA methylation (ASM) within disease-related loci has been proposed to be useful for the interpretation of GWAS results. To explore mechanisms that may underlie associations between Alzheimer’s disease (AD) and schizophrenia risk CLU gene and verbal memory, one of the most affected cognitive domains in both conditions, we studied DNA methylation in a region between AD-associated SNPs rs9331888 and rs9331896 in 72 healthy individuals and 73 schizophrenia patients. Using single-molecule real-time bisulfite sequencing we assessed the haplotype-dependent ASM in this region. We then investigated whether its methylation could influence episodic verbal memory measured with the Rey Auditory Verbal Learning Test in these two cohorts. The region showed a complex methylation pattern, which was similar in healthy and schizophrenia individuals and unrelated to haplotypes. The pattern predicted memory scores in controls. The results suggest that epigenetic modifications within the CLU locus may play a role in memory variation, independent of ASM. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

Glyphosate resistance and EPSPS gene duplication: Convergent evolution in multiple plant species.

One of the increasingly widespread mechanisms of resistance to the herbicide glyphosate is copy number variation (CNV) of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. EPSPS gene duplication has been reported in eight weed species, ranging from 3-5 extra copies to more than 150 extra copies. In the case of Palmer amaranth (Amaranthus palmeri), a section of >300 kb containing EPSPS and many other genes has been replicated and inserted at new loci throughout the genome, resulting in significant increase in total genome size. The replicated sequence contains several classes of mobile genetic elements including helitrons, raising the intriguing possibility of extra-chromosomal replication of the EPSPS-containing sequence. In kochia (Kochia scoparia), from three to more than 10 extra EPSPS copies are arranged as a tandem gene duplication at one locus. In the remaining six weed species that exhibit EPSPS gene duplication, little is known about the underlying mechanisms of gene duplication or their entire sequence. There is mounting evidence that adaptive gene amplification is an important mode of evolution in the face of intense human-mediated selection pressure. The convergent evolution of CNVs for glyphosate resistance in weeds, through at least two different mechanisms, may be indicative of a more general importance for this mechanism of adaptation in plants. CNVs warrant further investigation across plant functional genomics for adaptation to biotic and abiotic stresses, particularly for adaptive evolution on rapid time scales.© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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September 21, 2019

Discovery and genotyping of structural variation from long-read haploid genome sequence data.

In an effort to more fully understand the full spectrum of human genetic variation, we generated deep single-molecule, real-time (SMRT) sequencing data from two haploid human genomes. By using an assembly-based approach (SMRT-SV), we systematically assessed each genome independently for structural variants (SVs) and indels resolving the sequence structure of 461,553 genetic variants from 2 bp to 28 kbp in length. We find that >89% of these variants have been missed as part of analysis of the 1000 Genomes Project even after adjusting for more common variants (MAF > 1%). We estimate that this theoretical human diploid differs by as much as ~16 Mbp with respect to the human reference, with long-read sequencing data providing a fivefold increase in sensitivity for genetic variants ranging in size from 7 bp to 1 kbp compared with short-read sequence data. Although a large fraction of genetic variants were not detected by short-read approaches, once the alternate allele is sequence-resolved, we show that 61% of SVs can be genotyped in short-read sequence data sets with high accuracy. Uncoupling discovery from genotyping thus allows for the majority of this missed common variation to be genotyped in the human population. Interestingly, when we repeat SV detection on a pseudodiploid genome constructed in silico by merging the two haploids, we find that ~59% of the heterozygous SVs are no longer detected by SMRT-SV. These results indicate that haploid resolution of long-read sequencing data will significantly increase sensitivity of SV detection.© 2017 Huddleston et al.; Published by Cold Spring Harbor Laboratory Press.

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September 21, 2019

Identification of a novel RASD1 somatic mutation in a USP8-mutated corticotroph adenoma.

Cushing’s disease (CD) is caused by pituitary corticotroph adenomas that secrete excess adrenocorticotropic hormone (ACTH). In these tumors, somatic mutations in the gene USP8 have been identified as recurrent and pathogenic and are the sole known molecular driver for CD. Although other somatic mutations were reported in these studies, their contribution to the pathogenesis of CD remains unexplored. No molecular drivers have been established for a large proportion of CD cases and tumor heterogeneity has not yet been investigated using genomics methods. Also, even in USP8-mutant tumors, a possibility may exist of additional contributing mutations, following a paradigm from other neoplasm types where multiple somatic alterations contribute to neoplastic transformation. The current study utilizes whole-exome discovery sequencing on the Illumina platform, followed by targeted amplicon-validation sequencing on the Pacific Biosciences platform, to interrogate the somatic mutation landscape in a corticotroph adenoma resected from a CD patient. In this USP8-mutated tumor, we identified an interesting somatic mutation in the gene RASD1, which is a component of the corticotropin-releasing hormone receptor signaling system. This finding may provide insight into a novel mechanism involving loss of feedback control to the corticotropin-releasing hormone receptor and subsequent deregulation of ACTH production in corticotroph tumors.

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September 21, 2019

Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

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September 21, 2019

Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar Chardonnay.

Chardonnay is the basis of some of the world’s most iconic wines and its success is underpinned by a historic program of clonal selection. There are numerous clones of Chardonnay available that exhibit differences in key viticultural and oenological traits that have arisen from the accumulation of somatic mutations during centuries of asexual propagation. However, the genetic variation that underlies these differences remains largely unknown. To address this knowledge gap, a high-quality, diploid-phased Chardonnay genome assembly was produced from single-molecule real time sequencing, and combined with re-sequencing data from 15 different Chardonnay clones. There were 1620 markers identified that distinguish the 15 clones. These markers were reliably used for clonal identification of independently sourced genomic material, as well as in identifying a potential genetic basis for some clonal phenotypic differences. The predicted parentage of the Chardonnay haplomes was elucidated by mapping sequence data from the predicted parents of Chardonnay (Gouais blanc and Pinot noir) against the Chardonnay reference genome. This enabled the detection of instances of heterosis, with differentially-expanded gene families being inherited from the parents of Chardonnay. Most surprisingly however, the patterns of nucleotide variation present in the Chardonnay genome indicate that Pinot noir and Gouais blanc share an extremely high degree of kinship that has resulted in the Chardonnay genome displaying characteristics that are indicative of inbreeding.

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July 19, 2019

A window into third-generation sequencing.

First- and second-generation sequencing technologies have led the way in revolutionizing the field of genomics and beyond, motivating an astonishing number of scientific advances, including enabling a more complete understanding of whole genome sequences and the information encoded therein, a more complete characterization of the methylome and transcriptome and a better understanding of interactions between proteins and DNA. Nevertheless, there are sequencing applications and aspects of genome biology that are presently beyond the reach of current sequencing technologies, leaving fertile ground for additional innovation in this space. In this review, we describe a new generation of single-molecule sequencing technologies (third-generation sequencing) that is emerging to fill this space, with the potential for dramatically longer read lengths, shorter time to result and lower overall cost.

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