In a poster presented at AGBT 2017, Fritz Sedlazeck from Johns Hopkins University describes the comparison of genome assemblies produced using long-read PacBio sequencing and short-read sequencing with 10x Genomics…
Global transcript structure resolution of high gene density genomes through multi-platform data integration.
Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Short telomeres induce a DNA damage response, senescence, and apoptosis, thus maintaining telomere length equilibrium is essential for cell viability. Telomerase addition of telomere repeats is tightly regulated in cells. To probe pathways that regulate telomere addition, we developed the ADDIT assay to measure new telomere addition at a single telomere in vivo. Sequence analysis showed telomerase-specific addition of repeats onto a new telomere occurred in just 48 hr. Using the ADDIT assay, we found that ATM is required for addition of new repeats onto telomeres in mouse cells. Evaluation of bulk telomeres, in both human and mouse cells, showed that blocking ATM inhibited telomere elongation. Finally, the activation of ATM through the inhibition of PARP1 resulted in increased telomere elongation, supporting the central role of the ATM pathway in regulating telomere addition. Understanding this role of ATM may yield new areas for possible therapeutic intervention in telomere-mediated disease. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Nature repurposes proteins via evolutionary processes. Such adaptation can come at the expense of the original protein’s function, which is a trade-off of adaptation. We sought to examine other potential adaptive trade-offs. We measured the effect on ampicillin resistance of ~12,500 unique single amino acid mutants of the TEM-1, TEM-17, TEM-19, and TEM-15 ß-lactamase alleles, which constitute an adaptive path in the evolution of cefotaxime resistance. These protein fitness landscapes were compared and used to calculate epistatic interactions between these mutations and the two mutations in the pathway (E104K and G238S). This series of protein fitness landscapes provides a systematic, quantitative description of pairwise/tertiary intragenic epistasis involving adaptive mutations. We find that the frequency of mutations exhibiting epistasis increases along the evolutionary pathway. Adaptation moves the protein to a region in the fitness landscape characterized by decreased mutational robustness and increased ruggedness, as measured by fitness effects of mutations and epistatic interactions for TEM-1’s original function. This movement to such a “fitness territory” has evolutionary consequences and is an important adaptive trade-off and cost of adaptation. Our systematic study provides detailed insight into the relationships between mutation, protein structure, protein stability, and epistasis and quantitatively depicts the different costs inherent in the evolution of new functions. Copyright © 2016 Elsevier Ltd. All rights reserved.
Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.
A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat.
Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.© 2018 John Wiley & Sons Ltd.
Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
RelA mutant Enterococcus faecium with multiantibiotic tolerance arising in an immunocompromised host.
Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state.The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relA gene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci. Copyright © 2017 Honsa et al.
Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.
Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism’s physiology, metabolism, and pathogenesis in human disease.
In the basic fitness landscape metaphor for molecular evolution, evolutionary pathways are presumed to follow uphill steps of increasing fitness. How evolution can cross fitness valleys is an open question. One possibility is that environmental changes alter the fitness landscape such that low-fitness sequences reside on a hill in alternate environments. We experimentally test this hypothesis on the antibiotic resistance gene TEM-15 ß-lactamase by comparing four evolutionary strategies shaped by environmental changes. The strategy that included initial steps of selecting for low antibiotic resistance (negative selection) produced superior alleles compared with the other three strategies. We comprehensively examined possible evolutionary pathways leading to one such high-fitness allele and found that an initially deleterious mutation is key to the allele’s evolutionary history. This mutation is an initial gateway to an otherwise relatively inaccessible area of sequence space and participates in higher-order, positive epistasis with a number of neutral to slightly beneficial mutations. The ability of negative selection and environmental changes to provide access to novel fitness peaks has important implications for natural evolutionary mechanisms and applied directed evolution.
A time- and cost-effective strategy to sequence mammalian Y Chromosomes: an application to the de novo assembly of gorilla Y.
The mammalian Y Chromosome sequence, critical for studying male fertility and dispersal, is enriched in repeats and palindromes, and thus, is the most difficult component of the genome to assemble. Previously, expensive and labor-intensive BAC-based techniques were used to sequence the Y for a handful of mammalian species. Here, we present a much faster and more affordable strategy for sequencing and assembling mammalian Y Chromosomes of sufficient quality for most comparative genomics analyses and for conservation genetics applications. The strategy combines flow sorting, short- and long-read genome and transcriptome sequencing, and droplet digital PCR with novel and existing computational methods. It can be used to reconstruct sex chromosomes in a heterogametic sex of any species. We applied our strategy to produce a draft of the gorilla Y sequence. The resulting assembly allowed us to refine gene content, evaluate copy number of ampliconic gene families, locate species-specific palindromes, examine the repetitive element content, and produce sequence alignments with human and chimpanzee Y Chromosomes. Our results inform the evolution of the hominine (human, chimpanzee, and gorilla) Y Chromosomes. Surprisingly, we found the gorilla Y Chromosome to be similar to the human Y Chromosome, but not to the chimpanzee Y Chromosome. Moreover, we have utilized the assembled gorilla Y Chromosome sequence to design genetic markers for studying the male-specific dispersal of this endangered species. © 2016 Tomaszkiewicz et al.; Published by Cold Spring Harbor Laboratory Press.
Long single-molecule reads can resolve the complexity of the influenza virus composed of rare, closely related mutant variants
As a result of a high rate of mutations and recombination events, an RNA-virus exists as a heterogeneous “swarm” of mutant variants. The long read length offered by single-molecule sequencing technologies allows each mutant variant to be sequenced in a single pass. However, high error rate limits the ability to reconstruct heterogeneous viral population composed of rare, related mutant variants. In this paper, we present 2SNV, a method able to tolerate the high error-rate of the single-molecule protocol and reconstruct mutant variants. 2SNV uses linkage between single nucleotide variations to efficiently distinguish them from read errors. To benchmark the sensitivity of 2SNV, we performed a single-molecule sequencing experiment on a sample containing a titrated level of known viral mutant variants. Our method is able to accurately reconstruct clone with frequency of 0.2 % and distinguish clones that differed in only two nucleotides distantly located on the genome. 2SNV outperforms existing methods for full-length viral mutant reconstruction. The open source implementation of 2SNV is freely available for download at http://?alan.?cs.?gsu.?edu/?NGS/???q=?content/?2snv.