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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  |  

The genome of the tegu lizard Salvator merianae: combining Illumina, PacBio, and optical mapping data to generate a highly contiguous assembly.

Reptiles are a species-rich group with great phenotypic and life history diversity but are highly underrepresented among the vertebrate species with sequenced genomes.Here, we report a high-quality genome assembly of the tegu lizard, Salvator merianae, the first lacertoid with a sequenced genome. We combined 74X Illumina short-read, 29.8X Pacific Biosciences long-read, and optical mapping data to generate a high-quality assembly with a scaffold N50 value of 55.4 Mb. The contig N50 value of this assembly is 521 Kb, making it the most contiguous reptile assembly so far. We show that the tegu assembly has the highest completeness of coding genes and conserved non-exonic elements (CNEs) compared to other reptiles. Furthermore, the tegu assembly has the highest number of evolutionarily conserved CNE pairs, corroborating a high assembly contiguity in intergenic regions. As in other reptiles, long interspersed nuclear elements comprise the most abundant transposon class. We used transcriptomic data, homology- and de novo gene predictions to annotate 22,413 coding genes, of which 16,995 (76%) likely have human orthologs as inferred by CESAR-derived gene mappings. Finally, we generated a multiple genome alignment comprising 10 squamates and 7 other amniote species and identified conserved regions that are under evolutionary constraint. CNEs cover 38 Mb (1.8%) of the tegu genome, with 3.3 Mb in these elements being squamate specific. In contrast to placental mammal-specific CNEs, very few of these squamate-specific CNEs (<20 Kb) overlap transposons, highlighting a difference in how lineage-specific CNEs originated in these two clades.The tegu lizard genome together with the multiple genome alignment and comprehensive conserved element datasets provide a valuable resource for comparative genomic studies of reptiles and other amniotes.

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September 21, 2019  |  

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

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July 19, 2019  |  

The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms.

Molecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.We generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.Recent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.

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July 19, 2019  |  

Chromosomal-level assembly of the Asian seabass genome using long sequence reads and multi-layered scaffolding.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species’ native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

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July 19, 2019  |  

Chromosome-level assembly of Arabidopsis thaliana Ler reveals the extent of translocation and inversion polymorphisms.

Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ~3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.

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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.

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July 19, 2019  |  

Host genome integration and giant virus-induced reactivation of the virophage mavirus.

Endogenous viral elements are increasingly found in eukaryotic genomes, yet little is known about their origins, dynamics, or function. Here we provide a compelling example of a DNA virus that readily integrates into a eukaryotic genome where it acts as an inducible antiviral defence system. We found that the virophage mavirus, a parasite of the giant Cafeteria roenbergensis virus (CroV), integrates at multiple sites within the nuclear genome of the marine protozoan Cafeteria roenbergensis. The endogenous mavirus is structurally and genetically similar to eukaryotic DNA transposons and endogenous viruses of the Maverick/Polinton family. Provirophage genes are not constitutively expressed, but are specifically activated by superinfection with CroV, which induces the production of infectious mavirus particles. Virophages can inhibit the replication of mimivirus-like giant viruses and an anti-viral protective effect of provirophages on their hosts has been hypothesized. We find that provirophage-carrying cells are not directly protected from CroV; however, lysis of these cells releases infectious mavirus particles that are then able to suppress CroV replication and enhance host survival during subsequent rounds of infection. The microbial host-parasite interaction described here involves an altruistic aspect and suggests that giant-virus-induced activation of provirophages might be ecologically relevant in natural protist populations.

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July 19, 2019  |  

Antibody 10-1074 suppresses viremia in HIV-1-infected individuals.

Monoclonal antibody 10-1074 targets the V3 glycan supersite on the HIV-1 envelope (Env) protein. It is among the most potent anti-HIV-1 neutralizing antibodies isolated so far. Here we report on its safety and activity in 33 individuals who received a single intravenous infusion of the antibody. 10-1074 was well tolerated and had a half-life of 24.0 d in participants without HIV-1 infection and 12.8 d in individuals with HIV-1 infection. Thirteen individuals with viremia received the highest dose of 30 mg/kg 10-1074. Eleven of these participants were 10-1074-sensitive and showed a rapid decline in viremia by a mean of 1.52 log10 copies/ml. Virologic analysis revealed the emergence of multiple independent 10-1074-resistant viruses in the first weeks after infusion. Emerging escape variants were generally resistant to the related V3-specific antibody PGT121, but remained sensitive to antibodies targeting nonoverlapping epitopes, such as the anti-CD4-binding-site antibodies 3BNC117 and VRC01. The results demonstrate the safety and activity of 10-1074 in humans and support the idea that antibodies targeting the V3 glycan supersite might be useful for the treatment and prevention of HIV-1 infection.

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July 19, 2019  |  

The impact of third generation genomic technologies on plant genome assembly.

Since the introduction of next generation sequencing, plant genome assembly projects do not need to rely on dedicated research facilities or community-wide consortia anymore, even individual research groups can sequence and assemble the genomes they are interested in. However, such assemblies are typically not based on the entire breadth of genomic technologies including genetic and physical maps and their contiguities tend to be low compared to the full-length gold standard reference sequences. Recently emerging third generation genomic technologies like long-read sequencing or optical mapping promise to bridge this quality gap and enable simple and cost-effective solutions for chromosomal-level assemblies.

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July 19, 2019  |  

Gorilla MHC class I gene and sequence variation in a comparative context.

Comparisons of MHC gene content and diversity among closely related species can provide insights into the evolutionary mechanisms shaping immune system variation. After chimpanzees and bonobos, gorillas are humans’ closest living relatives; but in contrast, relatively little is known about the structure and variation of gorilla MHC class I genes (Gogo). Here, we combined long-range amplifications and long-read sequencing technology to analyze full-length MHC class I genes in 35 gorillas. We obtained 50 full-length genomic sequences corresponding to 15 Gogo-A alleles, 4 Gogo-Oko alleles, 21 Gogo-B alleles, and 10 Gogo-C alleles including 19 novel coding region sequences. We identified two previously undetected MHC class I genes related to Gogo-A and Gogo-B, respectively, thereby illustrating the potential of this approach for efficient and highly accurate MHC genotyping. Consistent with their phylogenetic position within the hominid family, individual gorilla MHC haplotypes share characteristics with humans and chimpanzees as well as orangutans suggesting a complex history of the MHC class I genes in humans and the great apes. However, the overall MHC class I diversity appears to be low further supporting the hypothesis that gorillas might have experienced a reduction of their MHC repertoire.

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July 19, 2019  |  

Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles.

The high-throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5′- to 3′-UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory – European Nucleotide Archive (EMBL-ENA) and the IPD-IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real-time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full-length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi-automated web service TypeLoader, to aid in the submission of sequences to the EMBL-ENA and the IPD-IMGT/HLA Database. In the IPD-IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full-length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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July 19, 2019  |  

TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton.

Transcription activator-like (TAL) effectors from Xanthomonas citri subsp. malvacearum (Xcm) are essential for bacterial blight of cotton (BBC). Here, by combining transcriptome profiling with TAL effector-binding element (EBE) prediction, we show that GhSWEET10, encoding a functional sucrose transporter, is induced by Avrb6, a TAL effector determining Xcm pathogenicity. Activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections. A BBC-resistant line carrying an unknown recessive b6 gene bears the same EBE as the susceptible line, but Avrb6-mediated induction of GhSWEET10 is reduced, suggesting a unique mechanism underlying b6-mediated resistance. We show via an extensive survey of GhSWEET transcriptional responsiveness to different Xcm field isolates that additional GhSWEETs may also be involved in BBC. These findings advance our understanding of the disease and resistance in cotton and may facilitate the development cotton with improved resistance to BBC.

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July 19, 2019  |  

Omics approaches to study gene regulatory networks for development in echinoderms.

Gene regulatory networks (GRNs) describe the interactions for a developmental process at a given time and space. Historically, perturbation experiments represent one of the key methods for analyzing and reconstructing a GRN, and the GRN governing early development in the sea urchin embryo stands as one of the more deeply dissected so far. As technology progresses, so do the methods used to address different biological questions. Next-generation sequencing (NGS) has become a standard experimental technique for genome and transcriptome sequencing and studies of protein-DNA interactions and DNA accessibility. While several efforts have been made toward the integration of different omics approaches for the study of the regulatory genome in many animals, in a few cases, these are applied with the purpose of reconstructing and experimentally testing developmental GRNs. Here, we review emerging approaches integrating multiple NGS technologies for the prediction and validation of gene interactions within echinoderm GRNs. These approaches can be applied to both ‘model’ and ‘non-model’ organisms. Although a number of issues still need to be addressed, advances in NGS applications, such as assay for transposase-accessible chromatin sequencing, combined with the availability of embryos belonging to different species, all separated by various evolutionary distances and accessible to experimental regulatory biology, place echinoderms in an unprecedented position for the reconstruction and evolutionary comparison of developmental GRNs. We conclude that sequencing technologies and integrated omics approaches allow the examination of GRNs on a genome-wide scale only if biological perturbation and cis-regulatory analyses are experimentally accessible, as in the case of echinoderm embryos.© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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July 19, 2019  |  

Single-molecule sequencing reveals the chromosome-scale genomic architecture of the nematode model organism Pristionchus pacificus.

The nematode Pristionchus pacificus is an established model for integrative evolutionary biology and comparative studies with Caenorhabditis elegans. While an existing genome draft facilitated the identification of several genes controlling various developmental processes, its high degree of fragmentation complicated virtually all genomic analyses. Here, we present a de novo genome assembly from single-molecule, long-read sequencing data consisting of 135 P. pacificus contigs. When combined with a genetic linkage map, 99% of the assembly could be ordered and oriented into six chromosomes. This allowed us to robustly characterize chromosomal patterns of gene density, repeat content, nucleotide diversity, linkage disequilibrium, and macrosynteny in P. pacificus. Despite widespread conservation of synteny between P. pacificus and C. elegans, we identified one major translocation from an autosome to the sex chromosome in the lineage leading to C. elegans. This highlights the potential of the chromosome-scale assembly for future genomic studies of P. pacificus. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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