Menu

Auto Tag: by SMRT

July 19, 2019  |  

The epigenomic landscape of prokaryotes.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

Read more
July 19, 2019  |  

Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5’CCCGA and 5’CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Read more
July 19, 2019  |  

A role for the bacterial GATC methylome in antibiotic stress survival.

Antibiotic resistance is an increasingly serious public health threat. Understanding pathways allowing bacteria to survive antibiotic stress may unveil new therapeutic targets. We explore the role of the bacterial epigenome in antibiotic stress survival using classical genetic tools and single-molecule real-time sequencing to characterize genomic methylation kinetics. We find that Escherichia coli survival under antibiotic pressure is severely compromised without adenine methylation at GATC sites. Although the adenine methylome remains stable during drug stress, without GATC methylation, methyl-dependent mismatch repair (MMR) is deleterious and, fueled by the drug-induced error-prone polymerase Pol IV, overwhelms cells with toxic DNA breaks. In multiple E. coli strains, including pathogenic and drug-resistant clinical isolates, DNA adenine methyltransferase deficiency potentiates antibiotics from the ß-lactam and quinolone classes. This work indicates that the GATC methylome provides structural support for bacterial survival during antibiotic stress and suggests targeting bacterial DNA methylation as a viable approach to enhancing antibiotic activity.

Read more
July 19, 2019  |  

DNA methylation on N(6)-adenine in mammalian embryonic stem cells.

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N(6)-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.

Read more
July 19, 2019  |  

An incomplete understanding of human genetic variation.

Deciphering the genetic basis of human disease requires a comprehensive knowledge of genetic variants irrespective of their class or frequency. Although an impressive number of human genetic variants have been catalogued, a large fraction of the genetic difference that distinguishes two human genomes is still not understood at the base-pair level. This is because the emphasis has been on single-nucleotide variation as opposed to less tractable and more complex genetic variants, including indels and structural variants. The latter, we propose, will have a large impact on human phenotypes but require a more systematic assessment of genomes at deeper coverage and alternate sequencing and mapping technologies. Copyright © 2016 by the Genetics Society of America.

Read more
July 19, 2019  |  

Comparative analyses of low, medium and high-resolution HLA typing technologies for human populations

Human Leukocyte Antigen (HLA) encoding genes are part of the major histocompatibility complex (MHC) on human chromosome 6. This region is one of the most polymorphic regions in the human genome. Prior knowledge of HLA allelic polymorphisms is clinically important for matching donor and recipient during organ/tissue transplantation. HLA allelic information is also useful in predicting immune responses to various infectious diseases, genetic disorders and autoimmune conditions. India harbors over a billion people and its population is untapped for HLA allelic diversity. In this study, we explored and compared three HLA typing methods for South Indian population, using Sequence-Specific Primers (SSP), NGS (Roche/454) and single- molecule sequencing (PacBio RS II) platforms. Over 1020 DNA samples were typed at low resolution using SSP method to determine the major HLA alleles within the South Indian population. These studies were followed up with medium resolution HLA typing of 80 samples based on exonic sequences on the Roche/454 sequencing system and high-resolution (6-8 digit) typing of 8 samples for HLA alleles of class I genes (HLA-A, B and C) and class II genes (HLA-DRB1 and DQB1) using PacBio RS II platform. The long reads delivered by SMRT technology, covered the full-length class I and class II genes/alleles in contiguous reads including untranslated regions, exons and introns, which provided phased SNP information. We have identified three novel alleles from PacBio data that were verified by Roche 454 sequencing. This is the first case study of HLA typing using second and third generation NGS technologies for an Indian population. The PacBio platform is a promising platform for large-scale HLA typing for establishing an HLA database for the untapped ethnic populations of India.

Read more
July 19, 2019  |  

AgIn: Measuring the landscape of CpG methylation of individual repetitive elements.

Determining the methylation state of regions with high copy numbers is challenging for second-generation sequencing, because the read length is insufficient to map reads uniquely, especially when repetitive regions are long and nearly identical to each other. Single-molecule real-time (SMRT) sequencing is a promising method for observing such regions, because it is not vulnerable to GC bias, it produces long read lengths, and its kinetic information is sensitive to DNA modifications.We propose a novel linear-time algorithm that combines the kinetic information for neighboring CpG sites and increases the confidence in identifying the methylation states of those sites. Using a practical read coverage of ~30-fold from an inbred strain medaka (Oryzias latipes), we observed that both the sensitivity and precision of our method on individual CpG sites were ~93.7%. We also observed a high correlation coefficient (R?=?0.884) between our method and bisulfite sequencing, and for 92.0% of CpG sites, methylation levels ranging over [0, 1] were in concordance within an acceptable difference 0.25. Using this method, we characterized the landscape of the methylation status of repetitive elements, such as LINEs, in the human genome, thereby revealing the strong correlation between CpG density and hypomethylation and detecting hypomethylation hot spots of LTRs and LINEs. We uncovered the methylation states for nearly identical active transposons, two novel LINE insertions of identity ~99% and length 6050 base pairs (bp) in the human genome, and 16 Tol2 elements of identity >99.8% and length 4682?bp in the medaka genome.AgIn (Aggregate on Intervals) is available at: https://github.com/hacone/AgIn CONTACT: ysuzuki@cb.k.u-tokyo.ac.jp, moris@cb.k.u-tokyo.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. © The Author(s) 2016. Published by Oxford University Press.

Read more
July 19, 2019  |  

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11?kb), single molecule, real-time sequencing.

The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed AT-richness [~80.6% (A?+?T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12?kb, with 50% of the reads between 15.5 and 50?kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [~90-99% (A?+?T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Read more
July 19, 2019  |  

High throughput random mutagenesis and Single Molecule Real Time Sequencing of the muscle nicotinic acetylcholine receptor.

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the a1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each a1 mutant was co-transfected with wildtype ß1, d, and e subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either a-bungarotoxin or tubocurarine. Eight a1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

Read more
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.

Read more
July 19, 2019  |  

Methylome analysis of two Xanthomonas spp. using Single-Molecule Real-Time Sequencing.

Single-molecule real-time (SMRT) sequencing allows identification of methylated DNA bases and methylation patterns/motifs at the genome level. Using SMRT sequencing, diverse bacterial methylomes including those of Helicobacter pylori, Lactobacillus spp., and Escherichia coli have been determined, and previously unreported DNA methylation motifs have been identified. However, the methylomes of Xanthomonas species, which belong to the most important plant pathogenic bacterial genus, have not been documented. Here, we report the methylomes of Xanthomonas axonopodis pv. glycines (Xag) strain 8ra and X. campestris pv. vesicatoria (Xcv) strain 85-10. We identified N(6)-methyladenine (6mA) and N(4)-methylcytosine (4mC) modification in both genomes. In addition, we assigned putative DNA methylation motifs including previously unreported methylation motifs via REBASE and MotifMaker, and compared methylation patterns in both species. Although Xag and Xcv belong to the same genus, their methylation patterns were dramatically different. The number of 4mC DNA bases in Xag (66,682) was significantly higher (29 fold) than in Xcv (2,321). In contrast, the number of 6mA DNA bases (4,147) in Xag was comparable to the number in Xcv (5,491). Strikingly, there were no common or shared motifs in the 10 most frequently methylated motifs of both strains, indicating they possess unique species- or strain-specific methylation motifs. Among the 20 most frequent motifs from both strains, for 9 motifs at least 1% of the methylated bases were located in putative promoter regions. Methylome analysis by SMRT sequencing technology is the first step toward understanding the biology and functions of DNA methylation in this genus.

Read more
July 19, 2019  |  

Examining sources of error in PCR by single-molecule sequencing.

Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification.

Read more
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.

Read more
July 19, 2019  |  

Comparative genomics reveals the diversity of restriction-modification systems and DNA methylation sites in Listeria monocytogenes.

Listeria monocytogenes is a bacterial pathogen that is found in a wide variety of anthropogenic and natural environments. Genome sequencing technologies are rapidly becoming a powerful tool in facilitating our understanding of how genotype, classification phenotypes, and virulence phenotypes interact to predict the health risks of individual bacterial isolates. Currently, 57 closed L. monocytogenes genomes are publicly available, representing three of the four phylogenetic lineages, and they suggest that L. monocytogenes has high genomic synteny. This study contributes an additional 15 closed L. monocytogenes genomes that were used to determine the associations between the genome and methylome with host invasion magnitude. In contrast to previous findings, large chromosomal inversions and rearrangements were detected in five isolates at the chromosome terminus and within rRNA genes, including a previously undescribed inversion within rRNA-encoding regions. Each isolate’s epigenome contained highly diverse methyltransferase recognition sites, even within the same serotype and methylation pattern. Eleven strains contained a single chromosomally encoded methyltransferase, one strain contained two methylation systems (one system on a plasmid), and three strains exhibited no methylation, despite the occurrence of methyltransferase genes. In three isolates a new, unknown DNA modification was observed in addition to diverse methylation patterns, accompanied by a novel methylation system. Neither chromosome rearrangement nor strain-specific patterns of epigenome modification observed within virulence genes were correlated with serotype designation, clonal complex, or in vitro infectivity. These data suggest that genome diversity is larger than previously considered in L. monocytogenes and that as more genomes are sequenced, additional structure and methylation novelty will be observed in this organism.Listeria monocytogenes is the causative agent of listeriosis, a disease which manifests as gastroenteritis, meningoencephalitis, and abortion. Among Salmonella, Escherichia coli, Campylobacter, and Listeria-causing the most prevalent foodborne illnesses-infection by L. monocytogenes carries the highest mortality rate. The ability of L. monocytogenes to regulate its response to various harsh environments enables its persistence and transmission. Small-scale comparisons of L. monocytogenes focusing solely on genome contents reveal a highly syntenic genome yet fail to address the observed diversity in phenotypic regulation. This study provides a large-scale comparison of 302 L. monocytogenes isolates, revealing the importance of the epigenome and restriction-modification systems as major determinants of L. monocytogenes phylogenetic grouping and subsequent phenotypic expression. Further examination of virulence genes of select outbreak strains reveals an unprecedented diversity in methylation statuses despite high degrees of genome conservation. Copyright © 2017 American Society for Microbiology.

Read more
July 19, 2019  |  

Single-molecule sequencing (PacBio) of the Staphylococcus capitis NRCS-A clone reveals the basis of multidrug resistance and adaptation to the Neonatal Intensive Care Unit environment.

The multi-resistant Staphylococcus capitis clone NRCS-A has recently been described as a major pathogen causing nosocomial, late-onset sepsis (LOS) in preterm neonates worldwide. NRCS-A representatives exhibit an atypical antibiotic resistance profile. Here, the complete closed genome (chromosomal and plasmid sequences) of NRCS-A prototype strain CR01 and the draft genomes of three other clinical NRCS-A strains from Australia, Belgium and the United Kingdom are annotated and compared to available non-NRCS-A S. capitis genomes. Our goal was to delineate the uniqueness of the NRCS-A clone with respect to antibiotic resistance, virulence factors and mobile genetic elements. We identified 6 antimicrobial resistance genes, all carried by mobile genetic elements. Previously described virulence genes present in the NRCS-A genomes are shared with the six non-NRCS-A S. capitis genomes. Overall, 63 genes are specific to the NRCS-A lineage, including 28 genes located in the methicillin-resistance cassette SCCmec. Among the 35 remaining genes, 25 are of unknown function, and 9 correspond to an additional type I restriction modification system (n = 3), a cytosine methylation operon (n = 2), and a cluster of genes related to the biosynthesis of teichoic acids (n = 4). Interestingly, a tenth gene corresponds to a resistance determinant for nisin (nsr gene), a bacteriocin secreted by potential NRCS-A strain niche competitors in the gut microbiota. The genomic characteristics presented here emphasize the contribution of mobile genetic elements to the emergence of multidrug resistance in the S. capitis NRCS-A clone. No NRCS-A-specific known virulence determinant was detected, which does not support a role for virulence as a driving force of NRCS-A emergence in NICUs worldwide. However, the presence of a nisin resistance determinant on the NRCS-A chromosome, but not in other S. capitis strains and most coagulase-negative representatives, might confer a competitive advantage to NRCS-A strains during the early steps of gut colonization in neonates. This suggests that the striking adaptation of NRCS-A to the NICU environment might be related to its specific antimicrobial resistance and also to a possible enhanced ability to challenge competing bacteria in its ecological niche.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.