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Asset Tag: PacBio

July 7, 2019

Direct detection and sequencing of damaged DNA bases.

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template – as a by-product of the sequencing method – through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

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July 7, 2019

Real-time sequencing.

This month’s Genome Watch describes the impact of next-generation sequencing on the ‘real-time’ analysis of pathogen genomes during outbreaks.

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July 7, 2019

Structural variation analysis with strobe reads.

Structural variation including deletions, duplications and rearrangements of DNA sequence are an important contributor to genome variation in many organisms. In human, many structural variants are found in complex and highly repetitive regions of the genome making their identification difficult. A new sequencing technology called strobe sequencing generates strobe reads containing multiple subreads from a single contiguous fragment of DNA. Strobe reads thus generalize the concept of paired reads, or mate pairs, that have been routinely used for structural variant detection. Strobe sequencing holds promise for unraveling complex variants that have been difficult to characterize with current sequencing technologies.We introduce an algorithm for identification of structural variants using strobe sequencing data. We consider strobe reads from a test genome that have multiple possible alignments to a reference genome due to sequencing errors and/or repetitive sequences in the reference. We formulate the combinatorial optimization problem of finding the minimum number of structural variants in the test genome that are consistent with these alignments. We solve this problem using an integer linear program. Using simulated strobe sequencing data, we show that our algorithm has better sensitivity and specificity than paired read approaches for structural variation identification.braphael@brown.edu

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July 7, 2019

Real-time tRNA transit on single translating ribosomes at codon resolution.

Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we use zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically relevant micromolar ligand concentrations. Translation at each codon is monitored by stable binding of transfer RNAs (tRNAs)-labelled with distinct fluorophores-to translating ribosomes, which allows direct detection of the identity of tRNA molecules bound to the ribosome and therefore the underlying messenger RNA (mRNA) sequence. We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule. Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

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July 7, 2019

Computational solutions to large-scale data management and analysis.

Today we can generate hundreds of gigabases of DNA and RNA sequencing data in a week for less than US$5,000. The astonishing rate of data generation by these low-cost, high-throughput technologies in genomics is being matched by that of other technologies, such as real-time imaging and mass spectrometry-based flow cytometry. Success in the life sciences will depend on our ability to properly interpret the large-scale, high-dimensional data sets that are generated by these technologies, which in turn requires us to adopt advances in informatics. Here we discuss how we can master the different types of computational environments that exist – such as cloud and heterogeneous computing – to successfully tackle our big data problems.

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July 7, 2019

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures.

Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of approximately 30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.

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July 7, 2019

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides.

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.

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July 7, 2019

Improved fabrication of zero-mode waveguides for single-molecule detection

Metallic subwavelength apertures can be used in epi-illumination fluorescence to achieve focal volume confinement. Because of the near field components inherent to small metallic structures, observation volumes are formed that are much smaller than the conventional diffraction limited volume attainable by high numerical aperture far field optics (circa a femtoliter). Observation volumes in the range of 10-4fl have been reported previously. Such apertures can be used for single-molecule detection at relatively high concentrations (up to 20µM) of fluorophores. Here, we present a novel fabrication of metallic subwavelength apertures in the visible range. Using a new electron beamlithography process, uniform arrays of such apertures can be manufactured efficiently in large numbers with diameters in the range of 60–100nm. The apertures were characterized by scanning electron microscopy, optical microscopy, focused ion beam cross sections/transmission electron microscopy, and fluorescence correlation spectroscopy measurements, which confirmed their geometry and optical confinement. Process throughput can be further increased using deep ultraviolet photolithography to replace electron beamlithography. This enables the production of aperture arrays in a high volume manufacturing environment.

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July 7, 2019

Complete mitogenome of Indian mottled eel, Anguilla bengalensis bengalensis (Gray, 1831) through PacBio RSII sequencing.

Complete mitogenome sequence for Anguilla bengalensis bengalensis (family Anguillidae) was generated through third-generation sequencing platform. The 16?714 bp mitgenome sequence contained 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a non-coding (control) region. The gene order was identical to that observed in most of the other vertebrates. The comparison of complete mitogenome sequence of Indian mottled eel generated during this study with two other subspecies did not agree with the taxonomic status of the three subspecies and considered as one species.

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July 7, 2019

Absence of genome reduction in diverse, facultative endohyphal bacteria.

Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.

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July 7, 2019

Accurate selfcorrection of errors in long reads using de Bruijn graphs.

New long read sequencing technologies, like PacBio SMRT and Oxford NanoPore, can produce sequencing reads up to 50,000 bp long but with an error rate of at least 15%. Reducing the error rate is necessary for subsequent utilisation of the reads in, e.g., de novo genome assembly. The error correction problem has been tackled either by aligning the long reads against each other or by a hybrid approach that uses the more accurate short reads produced by second generation sequencing technologies to correct the long reads.We present an error correction method that uses long reads only. The method consists of two phases: first we use an iterative alignment-free correction method based on de Bruijn graphs with increasing length of k-mers, and second, the corrected reads are further polished using long-distance dependencies that are found using multiple alignments. According to our experiments the proposed method is the most accurate one relying on long reads only for read sets with high coverage. Furthermore, when the coverage of the read set is at least 75x, the throughput of the new method is at least 20% higher.LoRMA is freely available at http://www.cs.helsinki.fi/u/lmsalmel/LoRMA/ CONTACT: leena.salmela@cs.helsinki.fi. © The Author(s) 2016. Published by Oxford University Press.

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July 7, 2019

Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing.

Difficulties in generating nuclear data for polyploids have impeded phylogenetic study of these groups. We describe a high-throughput protocol and an associated bioinformatics pipeline (Pipeline for Untangling Reticulate Complexes (Purc)) that is able to generate these data quickly and conveniently, and demonstrate its efficacy on accessions from the fern family Cystopteridaceae. We conclude with a demonstration of the downstream utility of these data by inferring a multi-labeled species tree for a subset of our accessions. We amplified four c. 1-kb-long nuclear loci and sequenced them in a parallel-tagged amplicon sequencing approach using the PacBio platform. Purc infers the final sequences from the raw reads via an iterative approach that corrects PCR and sequencing errors and removes PCR-mediated recombinant sequences (chimeras). We generated data for all gene copies (homeologs, paralogs, and segregating alleles) present in each of three sets of 50 mostly polyploid accessions, for four loci, in three PacBio runs (one run per set). From the raw sequencing reads, Purc was able to accurately infer the underlying sequences. This approach makes it easy and economical to study the phylogenetics of polyploids, and, in conjunction with recent analytical advances, facilitates investigation of broad patterns of polyploid evolution.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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July 7, 2019

Cultivation of a chemoautotroph from the SUP05 clade of marine bacteria that produces nitrite and consumes ammonium.

Marine oxygen minimum zones (OMZs) are expanding regions of intense nitrogen cycling. Up to half of the nitrogen available for marine organisms is removed from the ocean in these regions. Metagenomic studies have identified an abundant group of sulfur-oxidizing bacteria (SUP05) with the genetic potential for nitrogen cycling and loss in OMZs. However, SUP05 have defied cultivation and their physiology remains untested. We cultured, sequenced and tested the physiology of an isolate from the SUP05 clade. We describe a facultatively anaerobic sulfur-oxidizing chemolithoautotroph that produces nitrite and consumes ammonium under anaerobic conditions. Genetic evidence that closely related strains are abundant at nitrite maxima in OMZs suggests that sulfur-oxidizing chemoautotrophs from the SUP05 clade are a potential source of nitrite, fueling competing nitrogen removal processes in the ocean.

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July 7, 2019

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform.

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. This bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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