Nanopore Sequencing

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Stefan Prost - One of the best experts on this subject based on the ideXlab platform.

  • real time dna barcoding in a rainforest using Nanopore Sequencing opportunities for rapid biodiversity assessments and local capacity building
    GigaScience, 2018
    Co-Authors: Aaron Pomerantz, Nicolás Peñafiel, Alejandro Arteaga, Lucas Bustamante, Frank Pichardo, Luis A. Coloma, Stefan Prost, Cesar L Barrioamoros, David Salazarvalenzuela
    Abstract:

    BackgroundAdvancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Choco rainforest. We used portable equipment, including the MinION Nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field.FindingsWe demonstrate that Nanopore Sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnologica Indoamerica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger Sequencing, and with further multiplexing of samples, Nanopore Sequencing can become a cost-effective approach for rapid and portable DNA barcoding.ConclusionsOverall, we establish how mobile laboratories and Nanopore Sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

  • Real-time DNA barcoding in a remote rainforest using Nanopore Sequencing
    bioRxiv, 2017
    Co-Authors: Aaron Pomerantz, Nicolás Peñafiel, Alejandro Arteaga, Lucas Bustamante, Frank Pichardo, Luis A. Coloma, Cesar L Barrio-amoros, David Salazar-valenzuela, Stefan Prost
    Abstract:

    Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack, in one of the most imperiled biodiversity hotspots: the Ecuadorian Choco rainforest. We utilized portable equipment, including the MinION DNA sequencer (Oxford Nanopore Technologies) and miniPCR (miniPCR), to perform DNA extraction, PCR amplification and real-time DNA barcode Sequencing of reptile specimens in the field. We demonstrate that Nanopore Sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. In addition, we generated sequence information at Universidad Tecnologica Indoamerica in Quito for the recently re-discovered Jambato toad Atelopus ignescens, which was thought to be extinct for 28 years, a rare species of blind snake Trilepida guayaquilensis, and two undescribed species of Dipsas snakes. In this study we establish how mobile laboratories and Nanopore Sequencing can help to accelerate species identification in remote areas (especially for species that are difficult to diagnose based on characters of external morphology), be applied to local research facilities in developing countries, and rapidly generate information for species that are rare, endangered and undescribed, which can potentially aid in conservation efforts.

Onur Mutlu - One of the best experts on this subject based on the ideXlab platform.

  • Nanopore Sequencing technology and tools for genome assembly computational analysis of the current state bottlenecks and future directions
    Briefings in Bioinformatics, 2019
    Co-Authors: Damla Senol Cali, Saugata Ghose, Can Alkan, Onur Mutlu
    Abstract:

    Nanopore Sequencing technology has the potential to render other Sequencing technologies obsolete with its ability to generate long reads and provide portability. However, high error rates of the technology pose a challenge while generating accurate genome assemblies. The tools used for Nanopore sequence analysis are of critical importance, as they should overcome the high error rates of the technology. Our goal in this work is to comprehensively analyze current publicly available tools for Nanopore sequence analysis to understand their advantages, disadvantages and performance bottlenecks. It is important to understand where the current tools do not perform well to develop better tools. To this end, we (1) analyze the multiple steps and the associated tools in the genome assembly pipeline using Nanopore sequence data, and (2) provide guidelines for determining the appropriate tools for each step. Based on our analyses, we make four key observations: (1) the choice of the tool for basecalling plays a critical role in overcoming the high error rates of Nanopore Sequencing technology. (2) Read-to-read overlap finding tools, GraphMap and Minimap, perform similarly in terms of accuracy. However, Minimap has a lower memory usage, and it is faster than GraphMap. (3) There is a trade-off between accuracy and performance when deciding on the appropriate tool for the assembly step. The fast but less accurate assembler Miniasm can be used for quick initial assembly, and further polishing can be applied on top of it to increase the accuracy, which leads to faster overall assembly. (4) The state-of-the-art polishing tool, Racon, generates high-quality consensus sequences while providing a significant speedup over another polishing tool, Nanopolish. We analyze various combinations of different tools and expose the trade-offs between accuracy, performance, memory usage and scalability. We conclude that our observations can guide researchers and practitioners in making conscious and effective choices for each step of the genome assembly pipeline using Nanopore sequence data. Also, with the help of bottlenecks we have found, developers can improve the current tools or build new ones that are both accurate and fast, to overcome the high error rates of the Nanopore Sequencing technology.

  • Nanopore Sequencing Technology and Tools: Computational Analysis of the Current State, Bottlenecks, and Future Directions
    arXiv: Genomics, 2017
    Co-Authors: Damla Senol Cali, Saugata Ghose, Can Alkan, Onur Mutlu
    Abstract:

    Nanopore Sequencing technology has the potential to render other Sequencing technologies obsolete with its ability to generate long reads and provide portability. However, high error rates of the technology pose a challenge while generating accurate genome assemblies. The tools used for Nanopore sequence analysis are of critical importance as they should overcome the high error rates of the technology. Our goal in this work is to comprehensively analyze current publicly available tools for Nanopore sequence analysis to understand their advantages, disadvantages, and performance bottlenecks. It is important to understand where the current tools do not perform well to develop better tools. To this end, we 1) analyze the multiple steps and the associated tools in the genome assembly pipeline using Nanopore sequence data, and 2) provide guidelines for determining the appropriate tools for each step. We analyze various combinations of different tools and expose the tradeoffs between accuracy, performance, memory usage and scalability. We conclude that our observations can guide researchers and practitioners in making conscious and effective choices for each step of the genome assembly pipeline using Nanopore sequence data. Also, with the help of bottlenecks we have found, developers can improve the current tools or build new ones that are both accurate and fast, in order to overcome the high error rates of the Nanopore Sequencing technology.

Jared T Simpson - One of the best experts on this subject based on the ideXlab platform.

  • detecting dna cytosine methylation using Nanopore Sequencing
    Nature Methods, 2017
    Co-Authors: Rachael E Workman, Jared T Simpson, Philip C Zuzarte, Matei David, Lewis Jonathan Dursi, Winston Timp
    Abstract:

    A hidden Markov model (HMM)-based tool enables detection of 5-methylcytosine (5-mC) from single-molecule Nanopore-Sequencing data generated directly from human genomic DNA without chemical treatment.

  • nanocall an open source basecaller for oxford Nanopore Sequencing data
    Bioinformatics, 2017
    Co-Authors: Matei David, Jared T Simpson, Lewis Jonathan Dursi, Paul C Boutros
    Abstract:

    Motivation: The highly portable Oxford Nanopore MinION sequencer has enabled new applications of genome Sequencing directly in the field. However, the MinION currently relies on a cloud computing platform, Metrichor (metrichor.com), for translating locally generated Sequencing data into basecalls. Results: To allow offline and private analysis of MinION data, we created Nanocall. Nanocall is the first freely available, open-source basecaller for Oxford Nanopore Sequencing data and does not require an internet connection. Using R7.3 chemistry, on two E.coli and two human samples, with natural as well as PCR-amplified DNA, Nanocall reads have ∼68% identity, directly comparable to Metrichor ‘1D’ data. Further, Nanocall is efficient, processing ∼2500 Kbp of sequence per core hour using the fastest settings, and fully parallelized. Using a 4 core desktop computer, Nanocall could basecall a MinION Sequencing run in real time. Metrichor provides the ability to integrate the ‘1D’ Sequencing of template and complement strands of a single DNA molecule, and create a ‘2D’ read. Nanocall does not currently integrate this technology, and addition of this capability will be an important future development. In summary, Nanocall is the first open-source, freely available, off-line basecaller for Oxford Nanopore Sequencing data. Availability and Implementation: Nanocall is available at github.com/mateidavid/nanocall, released under the MIT license. Contact: matei.david@oicr.on.ca Supplementary information: Supplementary data are available at Bioinformatics online.

  • nanocall an open source basecaller for oxford Nanopore Sequencing data
    bioRxiv, 2016
    Co-Authors: Matei David, Lewis Jonathan Dursi, Paul C Boutros, Jared T Simpson
    Abstract:

    Motivation: The highly portable Oxford Nanopore MinION sequencer has enabled new applications of genome Sequencing directly in the field. However, the MinION currently relies on a cloud computing platform, Metrichor (metrichor.com), for translating locally generated Sequencing data into basecalls. Results: To allow offline and private analysis of MinION data, we created Nanocall. Nanocall is the first freely-available, open-source basecaller for Oxford Nanopore Sequencing data and does not require an internet connection. On two ecoli and two human samples, with natural as well as PCR-amplified DNA, Nanocall reads have ~68% identity, directly comparable to Metrichor "1D" data. Further, Nanocall is efficient, processing ~500Kbp of sequence per core hour, and fully parallelized. Using 8 cores, Nanocall could basecall a MinION Sequencing run in real time. Metrichor provides the ability to integrate the "1D" Sequencing of template and complement strands of a single DNA molecule, and create a "2D" read. Nanocall does not currently integrate this technology, and addition of this capability will be an important future development. In summary, Nanocall is the first open-source, freely available, off-line basecaller for Oxford Nanopore Sequencing data. Availability: Nanocall is available at github.com/mateidavid/nanocall, released under the MIT license. Contact: matei.david at oicr.on.ca

Herman P. Spaink - One of the best experts on this subject based on the ideXlab platform.

  • Author Correction: Rapid de novo assembly of the European eel genome from Nanopore Sequencing reads
    Scientific Reports, 2019
    Co-Authors: Hans J. Jansen, Michael Liem, Sylvie Dufour, Finnarne Weltzien, William Swinkels, Alex Koelewijn, A P Palstra, Bernd Pelster, Susanne Jong-raadsen, Herman P. Spaink
    Abstract:

    This Article contains errors. Since the publication of this Article, the website hosting the assembly data has become inactive. The data has now been re-deposited in the DataverseNO repository. As such, the corrected Data Availability section should be as follows. Data Availability The Nanopore Sequencing data are available in the European Nucleotide Archive (accession number PRJEB20018). The Racon- and Pilon-corrected candidate assembly is available at https://doi.org/10.18710/NMTGUN. The TULIP-scripts are available at https://github.com/Generade-nl.

  • rapid de novo assembly of the european eel genome from Nanopore Sequencing reads
    Scientific Reports, 2017
    Co-Authors: Hans J. Jansen, Michael Liem, Susanne Jongraadsen, Sylvie Dufour, Finnarne Weltzien, William Swinkels, Alex Koelewijn, A P Palstra, Bernd Pelster, Herman P. Spaink
    Abstract:

    We have sequenced the genome of the endangered European eel using the MinION by Oxford Nanopore, and assembled these data using a novel algorithm specifically designed for large eukaryotic genomes. For this 860 Mbp genome, the entire computational process takes two days on a single CPU. The resulting genome assembly significantly improves on a previous draft based on short reads only, both in terms of contiguity (N50 1.2 Mbp) and structural quality. This combination of affordable Nanopore Sequencing and light weight assembly promises to make high-quality genomic resources accessible for many non-model plants and animals.

  • De novo whole-genome assembly of a wild type yeast isolate using Nanopore Sequencing
    F1000Research, 2017
    Co-Authors: Michael Liem, Hans J. Jansen, Ron P. Dirks, Christiaan V. Henkel, G. Paul H. Van Heusden, Richard J.l.f. Lemmers, Trifa Omer, Shuai Shao, Peter J. Punt, Herman P. Spaink
    Abstract:

    Background : The introduction of the MinION Sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome Sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other Sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used Nanopore Sequencing as a tool to classify yeast strains. Methods : We compared various technical and software developments for the Nanopore Sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results : In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read Sequencing. This strain was originally named Candida vartiovaarae ( Torulopsis vartiovaarae ) based on ribosomal RNA Sequencing. We show that the assembly using Nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the Nanopore Sequencing protocol, showing that Nanopore-derived assemblies provide the highest contiguity. Conclusions : The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read Sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

Christopher E. Carr - One of the best experts on this subject based on the ideXlab platform.

  • Nanopore Sequencing at Mars, Europa, and Microgravity Conditions
    bioRxiv, 2019
    Co-Authors: Christopher E. Carr, Noelle C. Bryan
    Abstract:

    Nanopore Sequencing, as represented by Oxford Nanopore Technologies9 MinION, is a promising technology for in situ life detection and for microbial monitoring including in support of human space exploration, due to its small size, low mass (~100 g) and low power (~1W). Now ubiquitous on Earth and previously demonstrated on the International Space Station (ISS), Nanopore Sequencing involves translocation of DNA through a biological Nanopore on timescales of milliseconds per base. Nanopore Sequencing is now being done in both controlled lab settings as well as in diverse environments that include ground, air and space vehicles. Future space missions may also utilize Nanopore Sequencing in reduced gravity environments, such as in the search for life on Mars (Earth-relative gravito-inertial acceleration (GIA) g = 0.378), or at icy moons such as Europa (g = 0.134) or Enceladus (g = 0.012). We confirm the ability to sequence at Mars as well as near Europa or Lunar (g = 0.166) and lower g levels, demonstrate the functionality of updated chemistry and Sequencing protocols under parabolic flight, and reveal consistent performance across g level, during dynamic accelerations, and despite vibrations with significant power at translocation-relevant frequencies. Our work strengthens the use case for Nanopore Sequencing in dynamic environments on Earth and in space, including as part of the search for nucleic-acid based life beyond Earth.

  • carrierseq a sequence analysis workflow for low input Nanopore Sequencing
    BMC Bioinformatics, 2018
    Co-Authors: Angel Mojarro, Christopher E. Carr, Julie Hachey, Gary Ruvkun, Maria T Zuber
    Abstract:

    Background Long-read Nanopore Sequencing technology is of particular significance for taxonomic identification at or below the species level. For many environmental samples, the total extractable DNA is far below the current input requirements of Nanopore Sequencing, preventing “sample to sequence” metagenomics from low-biomass or recalcitrant samples.

  • carrierseq a sequence analysis workflow for low input Nanopore Sequencing
    bioRxiv, 2017
    Co-Authors: Angel Mojarro, Julie Hachey, Gary Ruvkun, Maria T Zuber, Christopher E. Carr
    Abstract:

    Motivation: Long-read Nanopore Sequencing technology is of particular significance for taxonomic identification at or below the species level. For many environmental samples, the total extractable DNA is far below the current input requirements of Nanopore Sequencing, preventing "sample to sequence" metagenomics from low-biomass or recalcitrant samples. Results: Here we address this problem by employing carrier Sequencing, a method to sequence low-input DNA by preparing the target DNA with a genomic carrier to achieve ideal library preparation and Sequencing stoichiometry without amplification. We then use CarrierSeq, a sequence analysis workflow to identify the low-input target reads from the genomic carrier. We tested CarrierSeq experimentally by Sequencing from a combination of 0.2 ng Bacillus subtilis ATCC 6633 DNA in a background of 1 μg Enterobacteria phage DNA. After filtering of carrier, low quality, and low complexity reads, we detected target reads (B. subtilis), contamination reads, and "high quality noise reads" (HQNRs) not mapping to the carrier, target or known lab contaminants. These reads appear to be artifacts of the Nanopore Sequencing process as they are associated with specific channels (pores). By treating reads as a Poisson arrival process, we implement a statistical test to reject data from channels dominated by HQNRs while retaining target reads. Availability: CarrierSeq is an open-source bash script with supporting python scripts which leverage a variety of bioinformatics software packages on macOS and Ubuntu. Supplemental documentation is available from Github - https://github.com/amojarro/carrierseq. In addition, we have complied all required dependencies in a Docker image available from - https://hub.docker.com/r/mojarro/carrierseq.

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