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Nanopore sequencing

Ruby White, Christophe Pellefigues, Franca Ronchese, Olivier Lamiable, David Eccles
Following a nanopore sequencing run of PCR products of three amplicons less than 1kb, an abundance of reads failed quality control due to template/complement mismatch. A BLAST search demonstrated that some of the failed reads mapped to two different genes -- an unexpected observation, given that PCR was carried out separately for each amplicon. A further investigation was carried out specifically to search for chimeric reads, using separate barcodes for each amplicon and trying two different ligation methods prior to sample loading...
2017: F1000Research
Nabeeh A Hasan, René L Warren, L Elaine Epperson, Allyson Malecha, David C Alexander, Christine Y Turenne, Daniel MacMillan, Inanc Birol, Stephen Pleasance, Robin Coope, Steven J M Jones, Marc G Romney, Monica Ng, Tracy Chan, Mabel Rodrigues, Patrick Tang, Jennifer L Gardy, Michael Strong
Mycobacterium chimaera, a nontuberculous mycobacterium (NTM) belonging to the Mycobacterium avium complex (MAC), is an opportunistic pathogen that can cause respiratory and disseminated disease. We report the complete genome sequence of a strain, SJ42, isolated from an immunocompromised male presenting with MAC pneumonia, assembled from Illumina and Oxford Nanopore data.
September 14, 2017: Genome Announcements
Lee J Kerkhof, Kevin P Dillon, Max M Häggblom, Lora R McGuinness
BACKGROUND: An approach utilizing the long-read capability of the Oxford Nanopore MinION to rapidly sequence bacterial ribosomal operons of complex natural communities was developed. Microbial fingerprinting employs domain-specific forward primers (16S rRNA subunit), reverse primers (23S rRNA subunit), and a high-fidelity Taq polymerase with proofreading capabilities. Amplicons contained both ribosomal subunits for broad-based phylogenetic assignment (~ 3900 bp of sequence), plus the intergenic spacer (ITS) region (~ 300 bp) for potential strain-specific identification...
September 15, 2017: Microbiome
Kazuo Imai, Norihito Tarumoto, Kazuhisa Misawa, Lucky Ronald Runtuwene, Jun Sakai, Kyoko Hayashida, Yuki Eshita, Ryuichiro Maeda, Josef Tuda, Takashi Murakami, Shigefumi Maesaki, Yutaka Suzuki, Junya Yamagishi, Takuya Maeda
BACKGROUND: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. METHODS: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P...
September 13, 2017: BMC Infectious Diseases
Joseph Larkin, Robert Y Henley, Vivek Jadhav, Jonas Korlach, Meni Wanunu
Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides-zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods...
September 11, 2017: Nature Nanotechnology
Anuj Nehra, Weizao Chen, Dimiter S Dimitrov, Anu Puri, Krishna Pal Singh
Solid-state nanopores within graphene-based materials are on the brink of fundamentally changing the sensing of desired bioanalytes through ion trafficking across nanoporous membranes. Here, we report on a two-electrode electrochemical biosensor comprised of a graphene oxide-polycarbonate track-etched nanosieve platform for the rapid and sensitive detection of the Human Immunodeficiency Virus Type 1 (HIV-1) envelope glycoprotein ectodomain (gp140MS). We have covalently linked an engineered high-affinity one-domain soluble CD4 fused to a human domain targeting HIV-1 coreceptor binding site and ferrocene (Fc) (2Dm2m) to the nanosieve platform...
September 13, 2017: ACS Applied Materials & Interfaces
Christopher M Austin, Mun Hua Tan, Katherine A Harrisson, Yin Peng Lee, Laurence J Croft, Paul Sunnucks, Alexandra Pavlova, Han Ming Gan
One of the most iconic Australian fish is the Murray cod, Maccullochella peelii (Mitchell 1838), a freshwater species that can grow to ∼1.8 metres in length and live to age ≥48 years. The Murray cod is of a conservation concern as a result of strong population contractions, but it is also popular for recreational fishing and is of growing aquaculture interest. In this study, we report the whole genome sequence of the Murray cod to support ongoing population genetics, conservation, and management research, as well as to better understand the evolutionary ecology and history of the species...
August 1, 2017: GigaScience
Jason L Weirather, Mariateresa de Cesare, Yunhao Wang, Paolo Piazza, Vittorio Sebastiano, Xiu-Jie Wang, David Buck, Kin Fai Au
Background: Given the demonstrated utility of Third Generation Sequencing [Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] long reads in many studies, a comprehensive analysis and comparison of their data quality and applications is in high demand. Methods: Based on the transcriptome sequencing data from human embryonic stem cells, we analyzed multiple data features of PacBio and ONT, including error pattern, length, mappability and technical improvements over previous platforms. We also evaluated their application to transcriptome analyses, such as isoform identification and quantification and characterization of transcriptome complexity, by comparing the performance of PacBio, ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina)...
2017: F1000Research
Benjamin Schwessinger, John P Rathjen
Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information...
2017: Methods in Molecular Biology
Nicholas A W Bell, Kaikai Chen, Sandip Ghosal, Maria Ricci, Ulrich F Keyser
In nanopore sensing, changes in ionic current are used to analyse single molecules in solution. The translocation dynamics of polyelectrolytes is of particular interest given potential applications such as DNA sequencing. In this paper, we determine how the dynamics of voltage driven DNA translocation can be affected by the nanopore geometry and hence the available configurational space for the DNA. Using the inherent geometrical asymmetry of a conically shaped nanopore, we examine how DNA dynamics depends on the directionality of transport...
August 30, 2017: Nature Communications
Jana Batovska, Stacey E Lynch, Brendan C Rodoni, Tim I Sawbridge, Noel Oi Cogan
With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2...
September 4, 2017: Journal of Virological Methods
Hai-Ling Liu, Jiao Cao, Sumaira Hanif, Chun-Ge Yuan, Jie Pang, Rastislav Levicky, Xing-Hua Xia, Kang Wang
Nanopore structures have been successfully employed in next generation DNA sequencing. For more complicated protein which normally contains 20 different amino acids, identifying the fluctuation of ionic current caused by different amino acids appears inadequate for protein sequencing. Therefore, it is highly desirable to develop size-controllable nanopores with optical activity that can provide additional structural information. Herein, we discovered the novel nanopore properties of the self-assembled ultramicroelectrodes originally developed by Bard and coworkers...
August 30, 2017: Analytical Chemistry
Sarah F Bailey, Melissa K Scheible, Christopher Williams, Deborah S B S Silva, Marina Hoggan, Christopher Eichman, Seth A Faith
Next-generation Sequencing (NGS) is a rapidly evolving technology with demonstrated benefits for forensic genetic applications, and the strategies to analyze and manage the massive NGS datasets are currently in development. Here, the computing, data storage, connectivity, and security resources of the Cloud were evaluated as a model for forensic laboratory systems that produce NGS data. A complete front-to-end Cloud system was developed to upload, process, and interpret raw NGS data using a web browser dashboard...
August 8, 2017: Forensic Science International. Genetics
Yuqian Zhang, Ankit Rana, Yiwen Stratton, Maria F Czyzyk-Krzeska, Leyla Esfandiari
MicroRNAs (miRs) are small noncoding RNAs that play a critical role in gene regulation. Recently, traces of cancer-related miRs have been identified in body fluids, which make them remarkable noninvasive biomarkers. In this study, a new nanopore-based detection scheme utilizing a borosilicate micropipette and an assay of complementary γ-peptide nucleic acid (γ-PNA) probes conjugated to polystyrene beads have been reported for the detection of miR-204 and miR-210 related to the clear cell Renal Cell Carcinoma (ccRCC)...
September 5, 2017: Analytical Chemistry
Rena Akahori, Itaru Yanagi, Yusuke Goto, Kunio Harada, Takahide Yokoi, Ken-Ichi Takeda
To achieve DNA sequencing with solid-state nanopores, the speed of the DNA in the nanopore must be controlled to obtain sequence-specific signals. In this study, we fabricated a nanopore-sensing system equipped with a DNA motion controller. DNA strands were immobilized on a Si probe, and approach of this probe to the nanopore vicinity could be controlled using a piezo actuator and stepper motor. The area of the Si probe was larger than the area of the membrane, which meant that the immobilized DNA could enter the nanopore without the need for the probe to scan to determine the location of the nanopore in the membrane...
August 22, 2017: Scientific Reports
Kaikai Chen, Matyas Juhasz, Felix Gularek, Elmar Weinhold, Yu Tian, Ulrich F Keyser, Nicholas A W Bell
Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method for determining the positions of short sequence motifs in double-stranded DNA molecules with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue we create covalently attached biotin labels at 5'-TCGA-3' sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to additional current blockade signals when the DNA passes through a conical quartz nanopore...
September 13, 2017: Nano Letters
Joe Parker, Andrew J Helmstetter, Dion Devey, Tim Wilkinson, Alexander S T Papadopulos
Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere. However, generation of datasets from large, complex genomes has been constrained to laboratories. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed...
August 21, 2017: Scientific Reports
Duah Alkam, Thidathip Wongsurawat, Piroon Jenjaroenpun, Skylar Connor, Charlotte Hobbs, Trudy M Wassenaar, Se-Ran Jun, Intawat Nookaew, David Ussery
We present here the complete genome sequences of three mumps virus (MuV) strains isolated from patients who tested positive for the mumps virus during a mumps outbreak in Springdale, AR (USA), in 2016. The virus genomes, sequenced with Oxford Nanopore technology, belong to genotype G and have an average length of 15,342 nucleotides (nt).
August 10, 2017: Genome Announcements
Jiwook Shim, Shouvik Banerjee, Hu Qiu, Kirby K H Smithe, David Estrada, Julian Bello, Eric Pop, Klaus Schulten, Rashid Bashir
Methylation at the 5-carbon position of the cytosine nucleotide base in DNA has been shown to be a reliable diagnostic biomarker for carcinogenesis. Early detection of methylation and intervention could drastically increase the effectiveness of therapy and reduce the cancer mortality rate. Current methods for detecting methylation involve bisulfite genomic sequencing, which are cumbersome and demand a large sample size of bodily fluids to yield accurate results. Hence, more efficient and cost effective methods are desired...
August 10, 2017: Nanoscale
Miten Jain, John R Tyson, Matthew Loose, Camilla L C Ip, David A Eccles, Justin O'Grady, Sunir Malla, Richard M Leggett, Ola Wallerman, Hans J Jansen, Vadim Zalunin, Ewan Birney, Bonnie L Brown, Terrance P Snutch, Hugh E Olsen
BACKGROUND: Long-read sequencing is rapidly evolving and reshaping the suite of opportunities for genomic analysis. For the MinION in particular, as both the platform and chemistry develop, the user community requires reference data to set performance expectations and maximally exploit third-generation sequencing. We performed an analysis of MinION data derived from whole genome sequencing of Escherichiacoli K-12 using the R9.0 chemistry, comparing the results with the older R7.3 chemistry...
2017: F1000Research
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