Menu

Auto Tag: ZMW

September 22, 2019  |  

Characterization and genomic analyses of Pseudomonas aeruginosa podovirus TC6: establishment of genus Pa11virus.

Phages have attracted a renewed interest as alternative to chemical antibiotics. Although the number of phages is 10-fold higher than that of bacteria, the number of genomically characterized phages is far less than that of bacteria. In this study, phage TC6, a novel lytic virus of Pseudomonas aeruginosa, was isolated and characterized. TC6 consists of an icosahedral head with a diameter of approximately 54 nm and a short tail with a length of about 17 nm, which are characteristics of the family Podoviridae. TC6 can lyse 86 out of 233 clinically isolated P. aeruginosa strains, thus showing application potentials for phage therapy. The linear double-stranded genomic DNA of TC6 consisted of 49796 base pairs and was predicted to contain 71 protein-coding genes. A total of 11 TC6 structural proteins were identified by mass spectrometry. Comparative analysis revealed that the P. aeruginosa phages TC6, O4, PA11, and IME180 shared high similarity at DNA sequence and proteome levels, among which PA11 was the first phage discovered and published. Meanwhile, these phages contain 54 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore proposed that these four phages can be classified as Pa11virus, comprising a new phage genus of Podoviridae that infects Pseudomonas spp. The results of this work promoted our understanding of phage biology, classification, and diversity.

Read more
September 22, 2019  |  

Real-time assembly of ribonucleoprotein complexes on nascent RNA transcripts.

Cellular protein-RNA complexes assemble on nascent transcripts, but methods to observe transcription and protein binding in real time and at physiological concentrations are not available. Here, we report a single-molecule approach based on zero-mode waveguides that simultaneously tracks transcription progress and the binding of ribosomal protein S15 to nascent RNA transcripts during early ribosome biogenesis. We observe stable binding of S15 to single RNAs immediately after transcription for the majority of the transcripts at 35?°C but for less than half at 20?°C. The remaining transcripts exhibit either rapid and transient binding or are unable to bind S15, likely due to RNA misfolding. Our work establishes the foundation for studying transcription and its coupled co-transcriptional processes, including RNA folding, ligand binding, and enzymatic activity such as in coupling of transcription to splicing, ribosome assembly or translation.

Read more
September 22, 2019  |  

Identification of DNA base modifications by means of Pacific Biosciences RS Sequencing technology.

Whole phage genomes can be sequenced readily using one or a combination of next generation sequencing (NGS) technologies. One of the most recently developed NGS platforms, the so-called Single-Molecule Real-Time (SMRT) sequencing approach provided by the PacBio RS platform, is particularly useful in providing complete (i.e., un-gapped) genome sequences, but differs from other technologies in that the platform also allows for downstream analysis to identify nucleotides that have been modified by DNA methylation. Here, we describe the methodological approach for the detection of genomic methylation motifs by means of SMRT sequencing.

Read more
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.

Read more
September 21, 2019  |  

DNA-guided delivery of single molecules into zero-mode waveguides.

Zero-mode waveguides (ZMWs) are powerful analytical tools corresponding to optical nanostructures fabricated in a thin metallic film capable of confining an excitation volume to the range of attoliters. This small volume of confinement allows single-molecule fluorescence experiments to be performed at physiologically relevant concentrations of fluorescently labeled biomolecules. Exactly one molecule to be studied must be attached at the floor of the ZMW for signal detection and analysis; however, the massive parallelism of these nanoarrays suffers from a Poissonian-limited distribution of these biomolecules. To date, there is no method available that provides full single-molecule occupancy of massively arrayed ZMWs. Here we report the performance of a DNA-guided method that uses steric exclusion properties of large DNA molecules to bias the Poissonian-limited delivery of single molecules. Non-Poissonian statistics were obtained with DNA molecules that contain a free-biotinylated extremity for efficient binding to the floor of the ZMW, which resulted in a decrease of accessibility for a second molecule. Both random-coiled and condensed DNA conformations drove non-Poissonian single-molecule delivery into ZMW arrays. The results suggest that an optimal balance between the rigidity and flexibility of the macromolecule is critical for favorable accessibility and single occupancy. The optimized method provides a means for full exploitation of these massively parallelized analytical tools.

Read more
September 21, 2019  |  

Real-time DNA sequencing from single polymerase molecules.

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

Read more
September 21, 2019  |  

Direct detection of DNA methylation during single-molecule, real-time sequencing.

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.

Read more
September 21, 2019  |  

A flexible and efficient template format for circular consensus sequencing and SNP detection.

A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region. In this article, we present a universal method for constructing these templates, as well as an application of their use. We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.

Read more
July 19, 2019  |  

Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology.

Many genomes have been sequenced to high-quality draft status using Sanger capillary electrophoresis and/or newer short-read sequence data and whole genome assembly techniques. However, even the best draft genomes contain gaps and other imperfections due to limitations in the input data and the techniques used to build draft assemblies. Sequencing biases, repetitive genomic features, genomic polymorphism, and other complicating factors all come together to make some regions difficult or impossible to assemble. Traditionally, draft genomes were upgraded to “phase 3 finished” status using time-consuming and expensive Sanger-based manual finishing processes. For more facile assembly and automated finishing of draft genomes, we present here an automated approach to finishing using long-reads from the Pacific Biosciences RS (PacBio) platform. Our algorithm and associated software tool, PBJelly, (publicly available at https://sourceforge.net/projects/pb-jelly/) automates the finishing process using long sequence reads in a reference-guided assembly process. PBJelly also provides “lift-over” co-ordinate tables to easily port existing annotations to the upgraded assembly. Using PBJelly and long PacBio reads, we upgraded the draft genome sequences of a simulated Drosophila melanogaster, the version 2 draft Drosophila pseudoobscura, an assembly of the Assemblathon 2.0 budgerigar dataset, and a preliminary assembly of the Sooty mangabey. With 24× mapped coverage of PacBio long-reads, we addressed 99% of gaps and were able to close 69% and improve 12% of all gaps in D. pseudoobscura. With 4× mapped coverage of PacBio long-reads we saw reads address 63% of gaps in our budgerigar assembly, of which 32% were closed and 63% improved. With 6.8× mapped coverage of mangabey PacBio long-reads we addressed 97% of gaps and closed 66% of addressed gaps and improved 19%. The accuracy of gap closure was validated by comparison to Sanger sequencing on gaps from the original D. pseudoobscura draft assembly and shown to be dependent on initial reference quality.

Read more
July 19, 2019  |  

Parallel confocal detection of single molecules in real time.

The confocal detection principle is extended to a highly parallel optical system that continuously analyzes thousands of concurrent sample locations. This is achieved through the use of a holographic laser illumination multiplexer combined with a confocal pinhole array before a prism dispersive element used to provide spectroscopic information from each confocal volume. The system is demonstrated to detect and identify single fluorescent molecules from each of several thousand independent confocal volumes in real time.

Read more
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.

Read more
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.

Read more
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.

Read more
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.

Read more
July 19, 2019  |  

Complex interplay among DNA modification, noncoding RNA expression and protein-coding RNA expression in Salvia miltiorrhiza chloroplast genome.

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box-like motif (CPGDMM1, "TATANNNATNA"), and an unknown motif (CPGDMM2 "WNYANTGAW"). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome.

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.