July 19, 2019  |  

Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing.

DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT) DNA sequencing that is capable of direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.


July 19, 2019  |  

Efficient and accurate whole genome assembly and methylome profiling of E. coli.

With the price of next generation sequencing steadily decreasing, bacterial genome assembly is now accessible to a wide range of researchers. It is therefore necessary to understand the best methods for generating a genome assembly, specifically, which combination of sequencing and bioinformatics strategies result in the most accurate assemblies. Here, we sequence three E. coli strains on the Illumina MiSeq, Life Technologies Ion Torrent PGM, and Pacific Biosciences RS. We then perform genome assemblies on all three datasets alone or in combination to determine the best methods for the assembly of bacterial genomes.Three E. coli strains – BL21(DE3), Bal225, and DH5a – were sequenced to a depth of 100× on the MiSeq and Ion Torrent machines and to at least 125× on the PacBio RS. Four assembly methods were examined and compared. The previously published BL21(DE3) genome [GenBank:AM946981.2], allowed us to evaluate the accuracy of each of the BL21(DE3) assemblies. BL21(DE3) PacBio-only assemblies resulted in a 90% reduction in contigs versus short read only assemblies, while N50 numbers increased by over 7-fold. Strikingly, the number of SNPs in PacBio-only assemblies were less than half that seen with short read assemblies (~20 SNPs vs. ~50 SNPs) and indels also saw dramatic reductions (~2 indel >5 bp in PacBio-only assemblies vs. ~12 for short-read only assemblies). Assemblies that used a mixture of PacBio and short read data generally fell in between these two extremes. Use of PacBio sequencing reads also allowed us to call covalent base modifications for the three strains. Each of the strains used here had a known covalent base modification genotype, which was confirmed by PacBio sequencing.Using data generated solely from the Pacific Biosciences RS, we were able to generate the most complete and accurate de novo assemblies of E. coli strains. We found that the addition of other sequencing technology data offered no improvements over use of PacBio data alone. In addition, the sequencing data from the PacBio RS allowed for sensitive and specific calling of covalent base modifications.


July 19, 2019  |  

The methylomes of six bacteria.

Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and C. jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N(6)-methyladenine ((m6)A) and N(4)-methylcytosine ((m4)C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases, it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTase genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. Two of these proved active. No attempt was made to detect 5-methylcytosine ((m5)C) recognition motifs from the SMRT® sequencing data because this modification produces weaker signals using current methods. However, all predicted (m6)A and (m4)C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active but also revealing their recognition sequences.


July 19, 2019  |  

The complex methylome of the human gastric pathogen Helicobacter pylori.

The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.


July 19, 2019  |  

A window into third-generation sequencing.

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


July 19, 2019  |  

Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution.

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N(6)-methyladenine (6 mA) and N(4)-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN(7)TAY-3’/3′-CTN(7)ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.


July 19, 2019  |  

Identification of restriction-modification systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and associated methylome analysis.

Bifidobacterium animalis subsp. lactis CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of B. animalis subsp. lactis CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in B. animalis subsp. lactis CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed.


July 19, 2019  |  

Returning to more finished genomes

Abstract Genomic data have become commonplace in most branches of the biological sciences and have fundamentally altered the way research is conducted. However, the predominance of short-read sequence data from second-generation sequencing technologies has commonly resulted in fragmented and partial genomic data characteristics. In this opinion, I will highlight how long, unbiased reads from single molecule, real-time (SMRT) sequencing now allow for a return to more contiguous and comprehensive views of genomes.


July 19, 2019  |  

Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1.

We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication.


July 19, 2019  |  

Entering the era of bacterial epigenomics with single molecule real time DNA sequencing.

DNA modifications, such as methylation guide numerous critical biological processes, yet epigenetic information has not routinely been collected as part of DNA sequence analyses. Recently, the development of single molecule real time (SMRT) DNA sequencing has enabled detection of modified nucleotides (e.g. 6mA, 4mC, 5mC) in parallel with acquisition of primary sequence data, based on analysis of the kinetics of DNA synthesis reactions. In bacteria, genome-wide mapping of methylated and unmethylated loci is now feasible. This technological advance sets the stage for comprehensive, mechanistic assessment of the effects of bacterial DNA methyltransferases (MTases)-which are ubiquitous, extremely diverse, and largely uncharacterized-on gene expression, chromosome structure, chromosome replication, and other fundamental biological processes. SMRT sequencing also enables detection of damaged DNA and has the potential to uncover novel DNA modifications. Copyright © 2013 Elsevier Ltd. All rights reserved.


July 19, 2019  |  

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome.After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51?N-6-methyladenines and 152?N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5a as a host bacterium to construct a shotgun library.This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.


July 19, 2019  |  

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.

The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.


July 19, 2019  |  

A random six-phase switch regulates pneumococcal virulence via global epigenetic changes.

Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.


July 19, 2019  |  

One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly.

Like a jigsaw puzzle with large pieces, a genome sequenced with long reads is easier to assemble. However, recent sequencing technologies have favored lowering per-base cost at the expense of read length. This has dramatically reduced sequencing cost, but resulted in fragmented assemblies, which negatively affect downstream analyses and hinder the creation of finished (gapless, high-quality) genomes. In contrast, emerging long-read sequencing technologies can now produce reads tens of kilobases in length, enabling the automated finishing of microbial genomes for under $1000. This promises to improve the quality of reference databases and facilitate new studies of chromosomal structure and variation. We present an overview of these new technologies and the methods used to assemble long reads into complete genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.


July 19, 2019  |  

qDNAmod: a statistical model-based tool to reveal intercellular heterogeneity of DNA modification from SMRT sequencing data.

In an isogenic cell population, phenotypic heterogeneity among individual cells is common and critical for survival of the population under different environment conditions. DNA modification is an important epigenetic factor that can regulate phenotypic heterogeneity. The single molecule real-time (SMRT) sequencing technology provides a unique platform for detecting a wide range of DNA modifications, including N6-methyladenine (6-mA), N4-methylcytosine (4-mC) and 5-methylcytosine (5-mC). Here we present qDNAmod, a novel bioinformatic tool for genome-wide quantitative profiling of intercellular heterogeneity of DNA modification from SMRT sequencing data. It is capable of estimating proportion of isogenic haploid cells, in which the same loci of the genome are differentially modified. We tested the reliability of qDNAmod with the SMRT sequencing data of Streptococcus pneumoniae strain ST556. qDNAmod detected extensive intercellular heterogeneity of DNA methylation (6-mA) in a clonal population of ST556. Subsequent biochemical analyses revealed that the recognition sequences of two type I restriction–modification (R-M) systems are responsible for the intercellular heterogeneity of DNA methylation initially identified by qDNAmod. qDNAmod thus represents a valuable tool for studying intercellular phenotypic heterogeneity from genome-wide DNA modification.


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.