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Nanopore sequencing

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https://www.readbyqxmd.com/read/28298449/real-time-sequencing-of-mycobacterium-tuberculosis-are-we-there-yet
#1
Robyn S Lee, Madhukar Pai
Whole genome sequencing has taken a leading role in epidemiologic studies of tuberculosis, but thus far, the real-time, clinical utility has been low, in part due to the requirement for culture. In Votintseva et al (J Clin Microbiol https://doi.org/10.1128/JCM.02483-16), authors present a new method for extracting Mycobacterium tuberculosis DNA directly from smear-positive respiratory samples, generating drug resistance predictions and phylogenetic trees in 44 hours using the Illumina MiSeq. Authors also illustrate the potential for < 24 hour turnaround time from DNA extraction to clinically-relevant results, with Illumina MiniSeq and Oxford Nanopore Technologies' MinION...
March 15, 2017: Journal of Clinical Microbiology
https://www.readbyqxmd.com/read/28298431/canu-scalable-and-accurate-long-read-assembly-via-adaptive-k-mer-weighting-and-repeat-separation
#2
Sergey Koren, Brian P Walenz, Konstantin Berlin, Jason R Miller, Nicholas H Bergman, Adam M Phillippy
Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8...
March 15, 2017: Genome Research
https://www.readbyqxmd.com/read/28288843/non-invasive-diagnosis-of-cutaneous-leishmaniasis-by-the-direct-boil-loop-mediated-isothermal-amplification-method-and-minion%C3%A2-nanopore-sequencing
#3
Kazuo Imai, Norihito Tarumoto, Kiyoko Amo, Makoto Takahashi, Naoya Sakamoto, Atsushi Kosaka, Yasuyuki Kato, Kei Mikita, Jun Sakai, Takashi Murakami, Yutaka Suzuki, Shigefumi Maesaki, Takuya Maeda
Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.
March 11, 2017: Parasitology International
https://www.readbyqxmd.com/read/28284619/identification-of-a-novel-species-of-papillomavirus-in-giraffe-lesions-using-nanopore-sequencing
#4
Bert Vanmechelen, Mads Frost Bertelsen, Annabel Rector, Joost J Van den Oord, Lies Laenen, Valentijn Vergote, Piet Maes
Papillomaviridae form a large family of viruses that are known to infect a variety of vertebrates, including mammals, reptiles, birds and fish. Infections usually give rise to minor skin lesions but can in some cases lead to the development of malignant neoplasia. In this study, we identified a novel species of papillomavirus (PV), isolated from warts of four giraffes (Giraffa camelopardalis). The sequence of the L1 gene was determined and found to be identical for all isolates. Using nanopore sequencing, the full sequence of the PV genome could be determined...
March 2017: Veterinary Microbiology
https://www.readbyqxmd.com/read/28275074/same-day-diagnostic-and-surveillance-data-for-tuberculosis-via-whole-genome-sequencing-of-direct-respiratory-samples
#5
Antonina A Votintseva, Phelim Bradley, Louise Pankhurst, Carlos Del Ojo Elias, Matthew Loose, Kayzad Nilgiriwala, Anirvan Chatterjee, E Grace Smith, Nicolas Sanderson, Timothy M Walker, Marcus R Morgan, David H Wyllie, A Sarah Walker, Tim E A Peto, Derrick W Crook, Zamin Iqbal
Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures)...
March 8, 2017: Journal of Clinical Microbiology
https://www.readbyqxmd.com/read/28272420/a-millisecond-micro-rna-separation-technique-by-a-hybrid-structure-of-nanopillars-and-nanoslits
#6
Qiong Wu, Noritada Kaji, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Kazuki Nagashima, Manabu Tokeshi, Tomoji Kawai, Yoshinobu Baba
A millisecond micro-RNA separation of a mixture of total RNA and genomic DNA, extracted from cultured HeLa cells, was successfully achieved using a hybrid structure of nanopillars and nanoslits contained inside a microchannel. The nanopillars, 250-nm in diameter and 100-nm in height, were fabricated with a 750-nm space inside the nanoslits, which were 100-nm in height and 25-μm in width; the nanopillars were then applied as a new sieve matrix. This ultra-fast technique for the separation of miRNA can be an effective pretreatment for semiconductor nanopore DNA sequencing, which has an optimum reading speed of 1 base/ms to obtain effective signal-to-noise ratio and discriminate each base by ion or tunneling current during the passage of nucleic acids...
March 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28269559/embedded-cmos-basecalling-for-nanopore-dna-sequencing
#7
Chengjie Wang, Junli Zheng, Sebastian Magierowski, Ebrahim Ghafar-Zadeh
DNA sequencing based on nanopore sensors is now entering the marketplace. The ability to interface this technology to established CMOS microelectronics promises significant improvements in functionality and miniaturization. Among the key functions to benefit from this interface will be basecalling, the conversion of raw electronic molecular signatures to nucleotide sequence predictions. This paper presents the design and performance potential of custom CMOS base-callers embedded alongside nanopore sensors. A basecalliing architecture implemented in 32-nm technology is discussed with the ability to process the equivalent of 20 human genomes per day in real-time at a power density of 5 W/cm2 assuming a 3-mer nanopore sensor...
August 2016: Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society
https://www.readbyqxmd.com/read/28251563/phosphate-modified-nucleotides-for-monitoring-enzyme-activity
#8
REVIEW
Susanne Ermert, Andreas Marx, Stephan M Hacker
Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins...
April 2017: Topics in Current Chemistry (Journal)
https://www.readbyqxmd.com/read/28227814/embedded-cmos-basecalling-for-nanopore-dna-sequencing
#9
Chengjie Wang, Junli Zheng, Sebastian Magierowski, Ebrahim Ghafar-Zadeh, Chengjie Wang, Junli Zheng, Sebastian Magierowski, Ebrahim Ghafar-Zadeh, Junli Zheng, Chengjie Wang, Ebrahim Ghafar-Zadeh, Sebastian Magierowski
DNA sequencing based on nanopore sensors is now entering the marketplace. The ability to interface this technology to established CMOS microelectronics promises significant improvements in functionality and miniaturization. Among the key functions to benefit from this interface will be basecalling, the conversion of raw electronic molecular signatures to nucleotide sequence predictions. This paper presents the design and performance potential of custom CMOS base-callers embedded alongside nanopore sensors. A basecalliing architecture implemented in 32-nm technology is discussed with the ability to process the equivalent of 20 human genomes per day in real-time at a power density of 5 W/cm(2) assuming a 3-mer nanopore sensor...
August 2016: Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society
https://www.readbyqxmd.com/read/28218898/detecting-dna-cytosine-methylation-using-nanopore-sequencing
#10
Jared T Simpson, Rachael E Workman, P C Zuzarte, Matei David, L J Dursi, Winston Timp
In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218897/mapping-dna-methylation-with-high-throughput-nanopore-sequencing
#11
Arthur C Rand, Miten Jain, Jordan M Eizenga, Audrey Musselman-Brown, Hugh E Olsen, Mark Akeson, Benedict Paten
DNA chemical modifications regulate genomic function. We present a framework for mapping cytosine and adenosine methylation with the Oxford Nanopore Technologies MinION using this nanopore sequencer's ionic current signal. We map three cytosine variants and two adenine variants. The results show that our model is sensitive enough to detect changes in genomic DNA methylation levels as a function of growth phase in Escherichia coli.
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218240/scaffolding-and-completing-genome-assemblies-in-real-time-with-nanopore-sequencing
#12
Minh Duc Cao, Son Hoang Nguyen, Devika Ganesamoorthy, Alysha G Elliott, Matthew A Cooper, Lachlan J M Coin
Third generation sequencing technologies provide the opportunity to improve genome assemblies by generating long reads spanning most repeat sequences. However, current analysis methods require substantial amounts of sequence data and computational resources to overcome the high error rates. Furthermore, they can only perform analysis after sequencing has completed, resulting in either over-sequencing, or in a low quality assembly due to under-sequencing. Here we present npScarf, which can scaffold and complete short read assemblies while the long read sequencing run is in progress...
February 20, 2017: Nature Communications
https://www.readbyqxmd.com/read/28208324/adsorption-induced-shape-transitions-in-bistable-nanopores-with-atomically-thin-walls
#13
Oleg E Shklyaev, Milton W Cole, Vincent H Crespi
Atomically thin cylindrical nanopores can change shape in response to physically adsorbed gas inside. Coupled to a gas reservoir, an initially collapsed pore can expand to allow the adsorbed gas to form concentric shells on the inner part of the pore, driven by adsorption energetics, not gas pressure. A lattice gas model describes the evolution of the nanotube pore shape and absorbed gas as a function of gas chemical potential at zero temperature. We found that narrow-enough tubes are always expanded and gas inside adsorbs in sequences of concentric shells as the gas chemical potential increases...
January 2017: Physical Review. E
https://www.readbyqxmd.com/read/28180136/sequencing-of-ebola-virus-genomes-using-nanopore-technology
#14
Thomas Hoenen
Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation sequencing methods, and was successfully used under outbreak conditions (Hoenen et al., 2016; Quick et al., 2016). Here we describe a protocol used for sequencing of Ebola virus under outbreak conditions using Nanopore technology, which we successfully implemented at the CDC/NIH diagnostic laboratory (de Wit et al...
November 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28177010/ultrabright-fluorescent-silica-particles-with-a-large-number-of-complex-spectra-excited-with-a-single-wavelength-for-multiplex-applications
#15
S Palantavida, B Peng, I Sokolov
We report on a novel approach to synthesize ultrabright fluorescent silica particles capable of producing a large number of complex spectra. The spectra can be excited using a single wavelength which is paramount in quantitative fluorescence imaging, flow cytometry and sensing applications. The approach employs the physical encapsulation of organic fluorescent molecules inside a nanoporous silica matrix with no dye leakage. As was recently demonstrated, such an encapsulation allowed for the encapsulation of very high concentrations of organic dyes without quenching their fluorescent efficiency...
February 8, 2017: Nanoscale
https://www.readbyqxmd.com/read/28166385/virus-hunting-discovery-of-new-episomal-circular-viruses-by-rolling-circle-techniques
#16
Bert Vanmechelen, Annabel Rector, Piet Maes
Many methods for the discovery of novel viruses are based on amplification of the virus using consensus or degenerate PCR primers. A downside of this approach is that it requires prior knowledge of the viral nucleotide sequence to be applicable. Presented in this unit is a method for the sequence-independent amplification of circular viral genomes that is based on the rolling-circle mechanism used by certain viruses in their natural replication cycle. The amplification of the virus of interest is coupled to the isolation of the viral genome by gel extraction following a restriction digestion...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28158244/dna-graphene-interactions-during-translocation-through-nanogaps
#17
Hiral N Patel, Ian Carroll, Rodolfo Lopez, Sandeep Sankararaman, Charles Etienne, Subba Ramaiah Kodigala, Mark R Paul, Henk W Ch Postma
We study how double-stranded DNA translocates through graphene nanogaps. Nanogaps are fabricated with a novel capillary-force induced graphene nanogap formation technique. DNA translocation signatures for nanogaps are qualitatively different from those obtained with circular nanopores, owing to the distinct shape of the gaps discussed here. Translocation time and conductance values vary by ∼ 100%, which we suggest are caused by local gap width variations. We also observe exponentially relaxing current traces...
2017: PloS One
https://www.readbyqxmd.com/read/28155888/forensic-snp-genotyping-using-nanopore-minion-sequencing
#18
Senne Cornelis, Yannick Gansemans, Lieselot Deleye, Dieter Deforce, Filip Van Nieuwerburgh
One of the latest developments in next generation sequencing is the Oxford Nanopore Technologies' (ONT) MinION nanopore sequencer. We studied the applicability of this system to perform forensic genotyping of the forensic female DNA standard 9947 A using the 52 SNP-plex assay developed by the SNPforID consortium. All but one of the loci were correctly genotyped. Several SNP loci were identified as problematic for correct and robust genotyping using nanopore sequencing. All these loci contained homopolymers in the sequence flanking the forensic SNP and most of them were already reported as problematic in studies using other sequencing technologies...
February 3, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28134443/challenges-and-opportunities-for-whole-genome-sequencing-based-surveillance-of-antibiotic-resistance
#19
Anita C Schürch, Willem van Schaik
Infections caused by drug-resistant bacteria are increasingly reported across the planet, and drug-resistant bacteria are recognized to be a major threat to public health and modern medicine. In this review, we discuss how whole-genome sequencing (WGS)-based approaches can contribute to the surveillance of the emergence and spread of antibiotic resistance. We outline the characteristics of sequencing technologies that are currently most used for WGS (Illumina short-read technologies and the long-read sequencing platforms developed by Pacific Biosciences and Oxford Nanopore)...
January 2017: Annals of the New York Academy of Sciences
https://www.readbyqxmd.com/read/28100585/fast-and-accurate-de-novo-genome-assembly-from-long-uncorrected-reads
#20
Robert Vaser, Ivan Sovic, Niranjan Nagarajan, Mile Sikic
The assembly of long reads from Pacific Biosciences and Oxford Nanopore Technologies typically requires resource intensive error correction and consensus generation steps to obtain high quality assemblies. We show that the error correction step can be omitted and high quality consensus sequences can be generated efficiently with a SIMD accelerated, partial order alignment based stand-alone consensus module called Racon. Based on tests with PacBio and Oxford Nanopore datasets we show that Racon coupled with Miniasm enables consensus genomes with similar or better quality than state-of-the-art methods while being an order of magnitude faster...
January 18, 2017: Genome Research
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