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September 21, 2019

Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

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July 19, 2019

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

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July 19, 2019

Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia.

Clostridium autoethanogenum strain JA1-1 (DSM 10061) is an acetogen capable of fermenting CO, CO2 and H2 (e.g. from syngas or waste gases) into biofuel ethanol and commodity chemicals such as 2,3-butanediol. A draft genome sequence consisting of 100 contigs has been published.A closed, high-quality genome sequence for C. autoethanogenum DSM10061 was generated using only the latest single-molecule DNA sequencing technology and without the need for manual finishing. It is assigned to the most complex genome classification based upon genome features such as repeats, prophage, nine copies of the rRNA gene operons. It has a low G + C content of 31.1%. Illumina, 454, Illumina/454 hybrid assemblies were generated and then compared to the draft and PacBio assemblies using summary statistics, CGAL, QUAST and REAPR bioinformatics tools and comparative genomic approaches. Assemblies based upon shorter read DNA technologies were confounded by the large number repeats and their size, which in the case of the rRNA gene operons were ~5 kb. CRISPR (Clustered Regularly Interspaced Short Paloindromic Repeats) systems among biotechnologically relevant Clostridia were classified and related to plasmid content and prophages. Potential associations between plasmid content and CRISPR systems may have implications for historical industrial scale Acetone-Butanol-Ethanol (ABE) fermentation failures and future large scale bacterial fermentations. While C. autoethanogenum contains an active CRISPR system, no such system is present in the closely related Clostridium ljungdahlii DSM 13528. A common prophage inserted into the Arg-tRNA shared between the strains suggests a common ancestor. However, C. ljungdahlii contains several additional putative prophages and it has more than double the amount of prophage DNA compared to C. autoethanogenum. Other differences include important metabolic genes for central metabolism (as an additional hydrogenase and the absence of a phophoenolpyruvate synthase) and substrate utilization pathway (mannose and aromatics utilization) that might explain phenotypic differences between C. autoethanogenum and C. ljungdahlii.Single molecule sequencing will be increasingly used to produce finished microbial genomes. The complete genome will facilitate comparative genomics and functional genomics and support future comparisons between Clostridia and studies that examine the evolution of plasmids, bacteriophage and CRISPR systems.

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July 19, 2019

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution.

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.

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July 19, 2019

Advantages of Single-Molecule Real-Time Sequencing in high-GC content genomes.

Next-generation sequencing has become the most widely used sequencing technology in genomics research, but it has inherent drawbacks when dealing with high-GC content genomes. Recently, single-molecule real-time sequencing technology (SMRT) was introduced as a third-generation sequencing strategy to compensate for this drawback. Here, we report that the unbiased and longer read length of SMRT sequencing markedly improved genome assembly with high GC content via gap filling and repeat resolution.

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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.

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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.

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July 19, 2019

A benchmark study on error assessment and quality control of CCS reads derived from the PacBio RS.

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.

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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.

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July 19, 2019

In vivo generation of DNA sequence diversity for cellular barcoding.

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic ‘barcode’ that can be recovered by DNA sequencing. Our method is a two-component system comprised of a genetic barcode cassette whose fragments are shuffled by Rci, a site-specific DNA invertase. The system is highly scalable, with the potential to generate theoretical diversities in the billions. We demonstrate the feasibility of this technique in Escherichia coli. Currently, this method could be employed to track the dynamics of populations of microbes through various bottlenecks. Advances of this method should prove useful in tracking interactions of cells within a network, and/or heterogeneity within complex biological samples.© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.

We have developed and validated an amplification-free method for generating DNA sequencing libraries from very low amounts of input DNA (500 picograms – 20 nanograms) for single- molecule sequencing on the Pacific Biosciences (PacBio) RS II sequencer. The common challenge of high input requirements for single-molecule sequencing is overcome by using a carrier DNA in conjunction with optimized sequencing preparation conditions and re-use of the MagBead-bound complex. Here we describe how this method can be used to produce sequencing yields comparable to those generated from standard input amounts, but by using 1000-fold less starting material.

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July 19, 2019

Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases.

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types.

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July 19, 2019

Chapter 20 – Real-time DNA sequencing from single polymerase molecules.

Pacific Biosciences has developed a method for real-time sequencing of single DNA molecules (Eid et al., 2009), with intrinsic sequencing rates of several bases per second and read lengths into the kilobase range. Conceptually, this sequencing approach is based on eavesdropping on the activity of DNA polymerase carrying out template-directed DNA polymerization. Performed in a highly parallel operational mode, sequential base additions catalyzed by each polymerase are detected with terminal phosphate-linked, fluorescence-labeled nucleotides. This chapter will first outline the principle of this single-molecule, real-time (SMRT) DNA sequencing method, followed by descriptions of its underlying components and typical sequencing run conditions. Two examples are provided which illustrate that, in addition to the DNA sequence, the dynamics of DNA polymerization from each enzyme molecules is directly accessible: the determination of base-specific kinetic parameters from single-molecule sequencing reads, and the characterization of DNA synthesis rate heterogeneities. Copyright 2010 Elsevier Inc. All rights reserved.

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July 19, 2019

Genome rearrangements and pervasive meiotic drive cause hybrid infertility in fission yeast.

Hybrid sterility is one of the earliest postzygotic isolating mechanisms to evolve between two recently diverged species. Here we identify causes underlying hybrid infertility of two recently diverged fission yeast species Schizosaccharomyces pombe and S. kambucha, which mate to form viable hybrid diploids that efficiently complete meiosis, but generate few viable gametes. We find that chromosomal rearrangements and related recombination defects are major but not sole causes of hybrid infertility. At least three distinct meiotic drive alleles, one on each S. kambucha chromosome, independently contribute to hybrid infertility by causing nonrandom spore death. Two of these driving loci are linked by a chromosomal translocation and thus constitute a novel type of paired meiotic drive complex. Our study reveals how quickly multiple barriers to fertility can arise. In addition, it provides further support for models in which genetic conflicts, such as those caused by meiotic drive alleles, can drive speciation.DOI: http://dx.doi.org/10.7554/eLife.02630.001. Copyright © 2014, Zanders et al.

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July 19, 2019

Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene.

The human fragile X mental retardation 1 (FMR1) gene contains a (CGG)(n) trinucleotide repeat in its 5′ untranslated region (5’UTR). Expansions of this repeat result in a number of clinical disorders with distinct molecular pathologies, including fragile X syndrome (FXS; full mutation range, greater than 200 CGG repeats) and fragile X-associated tremor/ataxia syndrome (FXTAS; premutation range, 55-200 repeats). Study of these diseases has been limited by an inability to sequence expanded CGG repeats, particularly in the full mutation range, with existing DNA sequencing technologies. Single-molecule, real-time (SMRT) sequencing provides an approach to sequencing that is fundamentally different from other “next-generation” sequencing platforms, and is well suited for long, repetitive DNA sequences. We report the first sequence data for expanded CGG-repeat FMR1 alleles in the full mutation range that reveal the confounding effects of CGG-repeat tracts on both cloning and PCR. A unique feature of SMRT sequencing is its ability to yield real-time information on the rates of nucleoside addition by the tethered DNA polymerase; for the CGG-repeat alleles, we find a strand-specific effect of CGG-repeat DNA on the interpulse distance. This kinetic signature reveals a novel aspect of the repeat element; namely, that the particular G bias within the CGG/CCG-repeat element influences polymerase activity in a manner that extends beyond simple nearest-neighbor effects. These observations provide a baseline for future kinetic studies of repeat elements, as well as for studies of epigenetic and other chemical modifications thereof.

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