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July 19, 2019

Living apart together: crosstalk between the core and supernumerary genomes in a fungal plant pathogen.

Eukaryotes display remarkable genome plasticity, which can include supernumerary chromosomes that differ markedly from the core chromosomes. Despite the widespread occurrence of supernumerary chromosomes in fungi, their origin, relation to the core genome and the reason for their divergent characteristics are still largely unknown. The complexity of genome assembly due to the presence of repetitive DNA partially accounts for this.Here we use single-molecule real-time (SMRT) sequencing to assemble the genome of a prominent fungal wheat pathogen, Fusarium poae, including at least one supernumerary chromosome. The core genome contains limited transposable elements (TEs) and no gene duplications, while the supernumerary genome holds up to 25 % TEs and multiple gene duplications. The core genome shows all hallmarks of repeat-induced point mutation (RIP), a defense mechanism against TEs, specific for fungi. The absence of RIP on the supernumerary genome accounts for the differences between the two (sub)genomes, and results in a functional crosstalk between them. The supernumerary genome is a reservoir for TEs that migrate to the core genome, and even large blocks of supernumerary sequence (>200 kb) have recently translocated to the core. Vice versa, the supernumerary genome acts as a refuge for genes that are duplicated from the core genome.For the first time, a mechanism was determined that explains the differences that exist between the core and supernumerary genome in fungi. Different biology rather than origin was shown to be responsible. A “living apart together” crosstalk exists between the core and supernumerary genome, accelerating chromosomal and organismal evolution.


July 19, 2019

Standardization and quality management in next-generation sequencing

DNA sequencing continues to evolve quickly even after > 30 years. Many new platforms suddenly appeared and former established systems have vanished in almost the same manner. Since establishment of next-generation sequencing devices, this progress gains momentum due to the continually growing demand for higher throughput, lower costs and better quality of data. In consequence of this rapid development, standardized procedures and data formats as well as comprehensive quality management considerations are still scarce. Here, we listed and summarized current standardization efforts and quality management initiatives from companies, organizations and societies in form of published studies and ongoing projects. These comprise on the one hand quality documentation issues like technical notes, accreditation checklists and guidelines for validation of sequencing workflows. On the other hand, general standard proposals and quality metrics are developed and applied to the sequencing workflow steps with the main focus on upstream processes. Finally, certain standard developments for downstream pipeline data handling, processing and storage are discussed in brief. These standardization approaches represent a first basis for continuing work in order to prospectively implement next-generation sequencing in important areas such as clinical diagnostics, where reliable results and fast processing is crucial. Additionally, these efforts will exert a decisive influence on traceability and reproducibility of sequence data.


July 19, 2019

Condition-dependent co-regulation of genomic clusters of virulence factors in the grapevine trunk pathogen Neofusicoccum parvum.

The ascomycete Neofusicoccum parvum, one of the causal agents of Botryosphaeria dieback, is a destructive wood-infecting fungus and a serious threat to grape production worldwide. The capability to colonize woody tissue, combined with the secretion of phytotoxic compounds, is thought to underlie its pathogenicity and virulence. Here, we describe the repertoire of virulence factors and their transcriptional dynamics as the fungus feeds on different substrates and colonizes the woody stem. We assembled and annotated a highly contiguous genome using single-molecule real-time DNA sequencing. Transcriptome profiling by RNA sequencing determined the genome-wide patterns of expression of virulence factors both in vitro (potato dextrose agar or medium amended with grape wood as substrate) and in planta. Pairwise statistical testing of differential expression, followed by co-expression network analysis, revealed that physically clustered genes coding for putative virulence functions were induced depending on the substrate or stage of plant infection. Co-expressed gene clusters were significantly enriched not only in genes associated with secondary metabolism, but also in those associated with cell wall degradation, suggesting that dynamic co-regulation of transcriptional networks contributes to multiple aspects of N. parvum virulence. In most of the co-expressed clusters, all genes shared at least a common motif in their promoter region, indicative of co-regulation by the same transcription factor. Co-expression analysis also identified chromatin regulators with correlated expression with inducible clusters of virulence factors, suggesting a complex, multi-layered regulation of the virulence repertoire of N. parvum.© 2016 BSPP AND JOHN WILEY & SONS LTD.


July 19, 2019

De novo assembly and phasing of a Korean human genome.

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9?Mb and a scaffold N50 size of 44.8?Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03?Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6?Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.


July 19, 2019

Short tandem repeats, segmental duplications, gene deletion, and genomic instability in a rapidly diversified immune gene family.

Genomic regions with repetitive sequences are considered unstable and prone to swift DNA diversification processes. A highly diverse immune gene family of the sea urchin (Strongylocentrotus purpuratus), called Sp185/333, is composed of clustered genes with similar sequence as well as several types of repeats ranging in size from short tandem repeats (STRs) to large segmental duplications. This repetitive structure may have been the basis for the incorrect assembly of this gene family in the sea urchin genome sequence. Consequently, we have resolved the structure of the family and profiled the members by sequencing selected BAC clones using Illumina and PacBio approaches.BAC insert assemblies identified 15 predicted genes that are organized into three clusters. Two of the gene clusters have almost identical flanking regions, suggesting that they may be non-matching allelic clusters residing at the same genomic locus. GA STRs surround all genes and appear in large stretches at locations of putatively deleted genes. GAT STRs are positioned at the edges of segmental duplications that include a subset of the genes. The unique locations of the STRs suggest their involvement in gene deletions and segmental duplications. Genomic profiling of the Sp185/333 gene diversity in 10 sea urchins shows that no gene repertoires are shared among individuals indicating a very high gene diversification rate for this family.The repetitive genomic structure of the Sp185/333 family that includes STRs in strategic locations may serve as platform for a controlled mechanism which regulates the processes of gene recombination, gene conversion, duplication and deletion. The outcome is genomic instability and allelic mismatches, which may further drive the swift diversification of the Sp185/333 gene family that may improve the immune fitness of the species.


July 19, 2019

The deep origin and recent loss of venom toxin genes in rattlesnakes.

The genetic origin of novel traits is a central but challenging puzzle in evolutionary biology. Among snakes, phospholipase A2 (PLA2)-related toxins have evolved in different lineages to function as potent neurotoxins, myotoxins, or hemotoxins. Here, we traced the genomic origin and evolution of PLA2 toxins by examining PLA2 gene number, organization, and expression in both neurotoxic and non-neurotoxic rattlesnakes. We found that even though most North American rattlesnakes do not produce neurotoxins, the genes of a specialized heterodimeric neurotoxin predate the origin of rattlesnakes and were present in their last common ancestor (~22 mya). The neurotoxin genes were then deleted independently in the lineages leading to the Western Diamondback (Crotalus atrox) and Eastern Diamondback (C. adamanteus) rattlesnakes (~6 mya), while a PLA2 myotoxin gene retained in C. atrox was deleted from the neurotoxic Mojave rattlesnake (C. scutulatus; ~4 mya). The rapid evolution of PLA2 gene number appears to be due to transposon invasion that provided a template for non-allelic homologous recombination. Copyright © 2016 Elsevier Ltd. All rights reserved.


July 19, 2019

Comparative DNA methylation and gene expression analysis identifies novel genes for structural congenital heart diseases.

For the majority of congenital heart diseases (CHDs), the full complexity of the causative molecular network, which is driven by genetic, epigenetic, and environmental factors, is yet to be elucidated. Epigenetic alterations are suggested to play a pivotal role in modulating the phenotypic expression of CHDs and their clinical course during life. Candidate approaches implied that DNA methylation might have a developmental role in CHD and contributes to the long-term progress of non-structural cardiac diseases. The aim of the present study is to define the postnatal epigenome of two common cardiac malformations, representing epigenetic memory, and adaption to hemodynamic alterations, which are jointly relevant for the disease course.We present the first analysis of genome-wide DNA methylation data obtained from myocardial biopsies of Tetralogy of Fallot (TOF) and ventricular septal defect patients. We defined stringent sets of differentially methylated regions between patients and controls, which are significantly enriched for genomic features like promoters, exons, and cardiac enhancers. For TOF, we linked DNA methylation with genome-wide expression data and found a significant overlap for hypermethylated promoters and down-regulated genes, and vice versa. We validated and replicated the methylation of selected CpGs and performed functional assays. We identified a hypermethylated novel developmental CpG island in the promoter of SCO2 and demonstrate its functional impact. Moreover, we discovered methylation changes co-localized with novel, differential splicing events among sarcomeric genes as well as transcription factor binding sites. Finally, we demonstrated the interaction of differentially methylated and expressed genes in TOF with mutated CHD genes in a molecular network.By interrogating DNA methylation and gene expression data, we identify two novel mechanism contributing to the phenotypic expression of CHDs: aberrant methylation of promoter CpG islands and methylation alterations leading to differential splicing. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.


July 19, 2019

Rapid functional and sequence differentiation of a tandemly repeated species-specific multigene family in Drosophila.

Gene clusters of recently duplicated genes are hotbeds for evolutionary change. However, our understanding of how mutational mechanisms and evolutionary forces shape the structural and functional evolution of these clusters is hindered by the high sequence identity among the copies, which typically results in their inaccurate representation in genome assemblies. The presumed testis-specific, chimeric gene Sdic originated, and tandemly expanded in Drosophila melanogaster, contributing to increased male-male competition. Using various types of massively parallel sequencing data, we studied the organization, sequence evolution, and functional attributes of the different Sdic copies. By leveraging long-read sequencing data, we uncovered both copy number and order differences from the currently accepted annotation for the Sdic region. Despite evidence for pervasive gene conversion affecting the Sdic copies, we also detected signatures of two episodes of diversifying selection, which have contributed to the evolution of a variety of C-termini and miRNA binding site compositions. Expression analyses involving RNA-seq datasets from 59 different biological conditions revealed distinctive expression breadths among the copies, with three copies being transcribed in females, opening the possibility to a sexually antagonistic effect. Phenotypic assays using Sdic knock-out strains indicated that should this antagonistic effect exist, it does not compromise female fertility. Our results strongly suggest that the genome consolidation of the Sdic gene cluster is more the result of a quick exploration of different paths of molecular tinkering by different copies than a mere dosage increase, which could be a recurrent evolutionary outcome in the presence of persistent sexual selection. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.


July 19, 2019

The genome of Chenopodium quinoa.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.


July 19, 2019

Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.


July 19, 2019

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ~400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.


July 19, 2019

Genomic structure of the horse major histocompatibility complex class II region resolved using PacBio long-read sequencing technology.

The mammalian Major Histocompatibility Complex (MHC) region contains several gene families characterized by highly polymorphic loci with extensive nucleotide diversity, copy number variation of paralogous genes, and long repetitive sequences. This structural complexity has made it difficult to construct a reliable reference sequence of the horse MHC region. In this study, we used long-read single molecule, real-time (SMRT) sequencing technology from Pacific Biosciences (PacBio) to sequence eight Bacterial Artificial Chromosome (BAC) clones spanning the horse MHC class II region. The final assembly resulted in a 1,165,328?bp continuous gap free sequence with 35 manually curated genomic loci of which 23 were considered to be functional and 12 to be pseudogenes. In comparison to the MHC class II region in other mammals, the corresponding region in horse shows extraordinary copy number variation and different relative location and directionality of the Eqca-DRB, -DQA, -DQB and -DOB loci. This is the first long-read sequence assembly of the horse MHC class II region with rigorous manual gene annotation, and it will serve as an important resource for association studies of immune-mediated equine diseases and for evolutionary analysis of genetic diversity in this region.


July 19, 2019

Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction.

The emergence of apomixis-the transition from sexual to asexual reproduction-is a prominent feature of modern citrus. Here we de novo sequenced and comprehensively studied the genomes of four representative citrus species. Additionally, we sequenced 100 accessions of primitive, wild and cultivated citrus. Comparative population analysis suggested that genomic regions harboring energy- and reproduction-associated genes are probably under selection in cultivated citrus. We also narrowed the genetic locus responsible for citrus polyembryony, a form of apomixis, to an 80-kb region containing 11 candidate genes. One of these, CitRWP, is expressed at higher levels in ovules of polyembryonic cultivars. We found a miniature inverted-repeat transposable element insertion in the promoter region of CitRWP that cosegregated with polyembryony. This study provides new insights into citrus apomixis and constitutes a promising resource for the mining of agriculturally important genes.


July 19, 2019

A new chicken genome assembly provides insight into avian genome structure.

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts. Copyright © 2017 Warren et al.


July 19, 2019

Widespread adenine N6-methylation of active genes in fungi.

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expression-associated epigenomic mark in eukaryotes.


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