Med. Nanopore sequencing significantly improves genome assembly of the protozoan parasite Trypanosoma cruzi. Nat. STAR: ultrafast universal RNA-seq aligner. NanoSatellite: accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION. The long reads ahead: de novo genome assembly using the MinION. Magi, A., Semeraro, R., Mingrino, A., Giusti, B. Answer: Fun question and it is of course not simple to answer. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. The technology relies on a nanoscale protein pore, or nanopore, that serves as a biosensor and is embedded in an electrically resistant polymer membrane1,3 (Fig. Lanfear, R., Schalamun, M., Kainer, D., Wang, W. & Schwessinger, B. MinIONQC: fast and simple quality control for MinION sequencing data. Similarly, multiple epigenetic features, including the endogenous 5mC methylome (at CpG sites), nucleosome occupancy and chromatin accessibility, can be simultaneously characterized on single long human DNA molecules by MeSMLR-seq (K.F.A., unpublished data, and ref. ONT developed new base callers as technology demonstrator software (for example, Nanonet, Scrappie and Flappie), which were subsequently implemented into the officially available software packages (for example, Albacore and Guppy). Gong, L. et al. Sci. 31). Proc. 39, 336346 (2020). A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification. Bioinformatics 35, 53725373 (2019). Nanopore sequencing is rapidly becoming one of the fastest and least expensive methods for deciphering genetic variations at the molecular level. Biol. This course will cover experimental considerations, sample collection and preparation theory, plus data analysis and visualisation. Kamath, G. M., Shomorony, I., Xia, F., Courtade, T. A. van Dijk, E. L., Jaszczyszyn, Y., Naquin, D. & Thermes, C. The third revolution in sequencing technology. Branton, D. et al. Fu, S. et al. PubMed NanoMethViz: an R/Bioconductor package for visualizing long-read methylation data. Brief. 17, 246253 (2016). Sci. Lorenz, D. A., Sathe, S., Einstein, J. M. & Yeo, G. W. Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution. Gigascience 8, giz043 (2019). 7, 146 (2018). 10, 579 (2019). MinIT is a data analysis device that eliminates the need for a computer to run MinION. Nat. Processive translocation of a DNA or RNA molecule leads to a characteristic current shift that is determined by multiple consecutive nucleotides (that is, k-mer) defined by the length of the nanopore sensing region1. 27, 472484 (2020). Unbiased strain-typing of arbovirus directly from mosquitoes using nanopore sequencing: a field-forward biosurveillance protocol. Article ONT devices take thousands of current measurements per second. In 2016 IEEE 6th International Conference on Computational Advances in Bio and Medical Sciences 16 (ICCABS, 2016). Preprint at bioRxiv https://doi.org/10.1101/2021.07.08.451578 (2021). The portable MinION device allows in-field and real-time genomic surveillance of emerging infectious diseases, aiding in phylogenetic and epidemiological investigations such as characterization of evolution rate, diagnostic targets, response to treatment and transmission rate. Nat. 3, 12411252 (2019). Establishment and cryptic transmission of Zika virus in Brazil and the Americas. Commun. PubMed Sci. Thank you for visiting nature.com. Merchant, C. A. et al. Translocation speed is controlled by a motor protein that ratchets the nucleic acid molecule through the nanopore in a step-wise manner. Derrington, I. M. et al. After removing the hairpin sequence, the template and complement reads, called the 1D reads, are used to generate a consensus sequence, called the 2D read, of higher accuracy. Nat. ONT also offers a direct cDNA sequencing protocol without PCR amplification, in contrast to many existing cDNA sequencing methods. MinION was also used to conduct genomic surveillance for Zika virus222, yellow fever virus223 and dengue virus224 outbreaks in Brazil. PLoS ONE 14, e0216471 (2019). Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing. & Yu, Y. W. Practical probabilistic and graphical formulations of long-read polyploid haplotype phasing. Rep. 7, 14521 (2017). 102) and deSALT103, for ONT transcriptome data. Bioinformatics 35, 29072915 (2019). K.F.A., Yunhao Wang, Y.Z. The width of each layer is proportional to the number of publications (in log2 scale). poRe: an R package for the visualization and analysis of nanopore sequencing data. Nature 554, 5055 (2018). Suzuki, A. et al. Huang, N., Nie, F., Ni, P., Luo, F. & Wang, J. SACall: a neural network basecaller for Oxford Nanopore sequencing data based on self-attention mechanism. Nanotechnol. A combination of Cas9-assisted target enrichment and ONT sequencing has characterized a 200-kb region spanning the breast cancer susceptibility gene BRCA1 and its flanking regions despite a high repetitive sequence fraction (>50%) and large gene size (~80kb)197. 1. Early versions of ONT sequencing used a 2D library preparation method to sequence each dsDNA molecule twice; the two strands of a dsDNA molecule are ligated together by a hairpin adapter, and a motor protein guides one strand (the template) through the nanopore, followed by the hairpin adapter and the second strand (the complement)40,41,42 (Fig. Jain, M., Olsen, H. E., Paten, B. Duke, J. L. et al. Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome. De novo yeast genome assemblies from MinION, PacBio and MiSeq platforms. Bioinformatics https://doi.org/10.1093/bioinformatics/btab540 (2021). 2d, solid line) for DNA sequencing through faster chemistry (increasing from ~30bases per s by R6 nanopore to ~450bases per s by R9.4 nanopore) and longer run times with the introduction of the Rev D ASIC chip. Preprint at bioRxiv https://doi.org/10.1101/073965 (2019). Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. & Leonardi, T. pycoQC, interactive quality control for Oxford Nanopore Sequencing. A typical sequencing experiment involves fragmentation of the genome into millions of molecules, which are size-selected and ligated to adapters.The set of fragments is referred to as a sequencing library, which is sequenced to produce a set of reads. Au, K. F., Underwood, J. G., Lee, L. & Wong, W. H. Improving PacBio long read accuracy by short read alignment. Recapturing and trapping single molecules with a solid-state nanopore. Faster sequencers, larger datasets, new challenges. Similarly, several other methods have combined various biochemical techniques with ONT sequencing (Table 1). Sci. Huang, Y. T., Liu, P. Y. Opportunities and challenges in long-read sequencing data analysis. Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads. PubMedGoogle Scholar. It should be noted, however, that in a rapidly evolving technology like nanopore sequencing, reviewing efforts can . Roach, N. P. et al. 4, bottom center, and Table 1). Update 05/04/2021: Oxford Nanopore Technologies has raised $272.5 million at a $3.44 billion post-money valuation ahead of an expected IPO this year. & DAurizio, R. Nanopore sequencing data analysis: state of the art, applications and challenges. Mol. Natl Acad. Mol. Fungus includes Candida auris, bacterium includes Salmonella, Neisseria meningitidis and Klebsiella pneumoniae and virus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, Zika, Venezuelan equine encephalitis, yellow fever, Lassa fever and dengue; HLA, human leukocyte antigens. Chem. PacBio recently announced its new HiFi sequencing method that it says is a new type of long-read sequencing technology allowing accuracy of 99.9% - on par with short reads and Sanger sequencing. Under the control of a motor protein, a double-stranded DNA (dsDNA) molecule (or an RNADNA hybrid duplex) is first unwound, then single-stranded DNA or RNA with negative charge is ratcheted through the nanopore, driven by the voltage. Genomics 111, 11081114 (2019). Currently, for DNA sequencing, ONT only supports the 1D method in which each strand of a dsDNA is ligated with an adapter and sequenced independently (Fig. Full-length HLA class I genotyping with the MinION nanopore sequencer. Biotechnol. Algorithms Mol. Google Scholar. A. & Quinlan, A. R. Poretools: a toolkit for analyzing nanopore sequence data. However, both of these approaches were limited in that each molecule could only be measured twice. In the same month, ONT announced the first nanopore sequencing device, MinION26. Biol. G3 8, 31313141 (2018). Microbes Infect. Nature 467, 190193 (2010). 17, 10971103 (2019). Direct detection of RNA modifications and structure using single molecule nanopore sequencing. David, M., Dursi, L. J., Yao, D., Boutros, P. C. & Simpson, J. T. Nanocall: an open source basecaller for Oxford Nanopore sequencing data. Genome Res. Brief. 10, 3359 (2019). Characterization of the human ESC transcriptome by hybrid sequencing. The detection of analytes and the sequencing of DNA using biological nanopores have seen major advances over recent years. Today, I'm going to talk about a different NGS platform that also allows real-time sequencing, but it uses a different principle. Parker, M. T. et al. Several advantages are offered by nanopore sequencing if it can be achieved. 17, e1009078 (2021). Ther. In the meantime, to ensure continued support, we are displaying the site without styles Beyond optimizing the nanopore and motor protein, several strategies have been developed to improve accuracy. However, overrepresented small fragments outside the desired size distribution may decrease sequencing yield because of higher efficiencies of both adapter ligation and translocation through nanopores than long fragments. 20, 237 (2019). Sci. Genome Biol. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. Georgieva, D., Liu, Q., Wang, K. & Egli, D. Detection of base analogs incorporated during DNA replication by nanopore sequencing. The full-length transcriptome of C. elegans using direct RNA sequencing. In this work, we show that . Nat. With ONT data alone, there remain drawbacks in estimating gene/isoform abundance, detecting splice sites and mapping alternative polyadenylation sites, although recent improvements in accuracy and throughput have advanced these analyses. Morisse, P., Lecroq, T. & Lefebvre, A. The long read length, portability and direct RNA sequencing capability of ONT devices have supported a diverse range of applications (Fig. The PromethION 2 devices are designed to be compact and accessible, utilising 2 PromethION Flow Cells that can generate hundreds of gigabases of data each. Sci. Microbiol. Sedlazeck, F. J. et al. The ability to directly detect DNA modifications using ONT data has enabled the simultaneous capture of genomic (that is, copy number variation) and epigenomic (that is, 5mC) alterations using only ONT data from brain tumor samples193. Jain, M. et al. 22, 95 (2021). To remove overrepresented small DNA fragments, various size selection methods (for example, the gel-based BluePippin system of Sage Science, magnetic beads and the Short Read Eliminator kit of Circulomics) have been used to obtain the desired data distribution and/or improve sequencing yield (Fig. In parallel, accuracy has been improved through new base-calling algorithms, including many developed through independent research32,44 (see below). 7 (World Scientific Press, 2019). Many applications combine long reads and short reads in the bioinformatics analyses, termed hybrid sequencing. Haplotype threading: accurate polyploid phasing from long reads. The MinION is a portable sequencer that can generate up to 50 Gb of data per Flow Cell. Runtuwene, L. R., Tuda, J. S. B., Mongan, A. E. & Suzuki, Y. 30, 693700 (2012). USA 105, 2064720652 (2008). 16, 114 (2015). Learn more on the advantages of real-time sequencing, Find out more about the Ultra-long sequencing kit. USA 106, 77027707 (2009). We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T . Amin, M. R., Skiena, S. & Schatz, M. C. NanoBLASTer: fast alignment and characterization of Oxford Nanopore single molecule sequencing reads. The analysis of proteins and peptides with nanopores, however, is complicated by the complex physicochemical structure of polypeptides and the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores. Natl Acad. The authors apologize to colleagues whose studies were not cited due to length and reference constraints. Hou, L. & Wang, Y. DEEP-LONG: a fast and accurate aligner for long RNA-seq. 22, 182 (2021). Cell 181, 914921 (2020). 34, 518524 (2016). Cartolano, M., Huettel, B., Hartwig, B., Reinhardt, R. & Schneeberger, K. cDNA library enrichment of full length transcripts for SMRT long read sequencing. Ip, C. L. C. et al. What are the disadvantages of nanopore sequencing? Rep. 8, 14408 (2018). Liu, Q. et al. Mapping DNA replication with nanopore sequencing. 49, 556559 (2010). Sanderson, N. D. et al. J. Exp. Structural, physiological and regulatory analysis of maltose transporter genes in Saccharomyces eubayanus CBS 12357T. Aw, J. G. A. et al. Article Yunhao Wang and A.B. 11, 96 (2016). Front. Rep. 7, 7213 (2017). However, the R9.4 and R9.5 have difficulty sequencing very long homopolymer runs because the current signal of CsgG is determined by approximately five consecutive nucleotides. Here, we focus on the current advantages as well as the limitations of the ONT nanopore sequencer, reviewing research publications available thus far. Liu, Q. et al. B. R. et al. Gigascience 9, giaa101 (2020). Nat Biotechnol 39, 13481365 (2021). Singh, M. et al. Genet. AERON: transcript quantification and gene-fusion detection using long reads. Nat. Identification and characterization of occult human-specific LINE-1 insertions using long-read sequencing technology. Nanopore sensing is a direct electronic analysis of the target molecule, Sequencing native DNA (and RNA) avoids bias introduced by amplification steps, opening up opportunities for epigenetic analysis, Workflows are kept simple by avoiding amplification or light-based measurement steps. For instance, ONT data alone were used to assemble the first genome of Rhizoctonia solani (a pathogenic fungal species that causes damping-off diseases in a wide range of crops)142, and hybrid sequencing data (ONT plus Illumina) were used to assemble the first draft genomes of Maccullochella peelii (Australias largest freshwater fish)143 and Amphiprion ocellaris (the common clown fish)144. 43, e116 (2015). Preprint at bioRxiv https://doi.org/10.1101/2020.05.18.101402 (2020). . More robust and user-friendly bioinformatics software, such as cloud storage and computing and real-time analysis, will provide a further boost to ONT sequencing applications, ultimately moving the technology beyond the lab and into daily life. Biotechnol. Diaz-Viraque, F. et al. In addition to the genome-polishing software Nanopolish109, ONT released Medaka, a neural network-based method, aiming for improved accuracy and speed compared to Nanopolish (Table 1). Bioinformatics 35, 2027 (2019). In Proc. Proc. Genome Biol. 4, 1236 (2019). 5). Cancer Biol. Proc. More recently, there has been considerable growth in alignment methods for high-throughput accurate short reads (for example, Illumina sequencing data) in response to the growth in next-generation sequencing. Methods 10, 563569 (2013). The fast5 format organizes the multidimensional data in a nested manner, allowing the piece-wise access/extraction of information of interest without navigating through the whole dataset. G3 10, 11931196 (2020). Preprint at medRxiv https://doi.org/10.1101/2020.03.05.20032011 (2020). Moore, S. C. et al. Single-molecule long-read sequencing reveals the chromatin basis of gene expression. Science 303, 11891192 (2004). Nucleobase recognition in ssDNA at the central constriction of the -hemolysin pore. & Coin, L. J. Realtime analysis and visualization of MinION sequencing data with npReader. In addition to sequencing length and accuracy, throughput is another important consideration for ONT sequencing applications. In recent years long-read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Many of these, such as tools for error correction, assembly and alignment, were developed for PacBio data but are also applicable to ONT data (Table 1). Sci. More recently, ONT direct RNA sequencing has made transcriptome-wide investigation of native RNA molecules feasible52,130,131. Accurate detection of m6A RNA modifications in native RNA sequences. Preprint at bioRxiv https://doi.org/10.1101/2021.01.22.427687 (2021). The membrane is resistant to the electric current, meaning the only way for current to flow across the membrane is to travel through the pore. BMC Bioinformatics 19, 50 (2018). BMC Genomics 19, 714 (2018). Tree Lab: portable genomics for early detection of plant viruses and pests in sub-Saharan Africa. 48, 11461163 (2020). USA 115, 97269731 (2018). 20, 129 (2019). Preprint at bioRxiv https://doi.org/10.1101/133058 (2017). Li, H. Minimap2: pairwise alignment for nucleotide sequences. A comprehensive examination of the feasibility of ONT cDNA sequencing (with R7 and R9 nanopores) in transcriptome analyses demonstrated its similar performance in gene isoform identification to PacBio long reads, both of which are superior to Illumina short reads42. 2, 148 (2019). Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Gong, L., Wong, C. H., Idol, J., Ngan, C. Y. Compared to PacBio, ONT performs better in detecting 5mC but has lower accuracy in detecting 6mA68,75,81. Preprint at bioRxiv https://doi.org/10.1101/2021.07.22.453313 (2021). With the increasing throughput of ONT sequencing, real-time surveillance has been applied to pathogens with larger genomes over the years, ranging from viruses of a few kilobases (for example, Ebola virus220, 1819kb; Zika virus222, 11kb; Venezuelan equine encephalitis virus225, 11.4kb; Lassa fever virus226, 10.4kb and SARS-CoV-2 coronavirus151, 29.8kb) to bacteria of several megabases (for example, Salmonella221, 5Mb; N. meningitidis227, 2Mb and K. pneumoniae228, 5.4Mb) and to human fungal pathogens with genomes of >10Mb (for example, Candida auris229, 12Mb). Bioinformatics 32, 764766 (2016). Biol. Bioinformatics 33, 4955 (2017). This third-generation technology is developed and marketed by Oxford Nanopore Technologies (ONT), that uses a small portable sequencing device called MinION [ 1 ]. Genome Biol. 56, 265270 (2019). Various approaches for extracting and purifying high-molecular-weight (HMW) DNA have been reported or applied to ONT sequencing, including spin columns (for example, Monarch Genomic DNA Purification kit, New England Biolabs), gravity-flow columns (for example, NucleoBond HMW DNA kit, Takara Bio), magnetic beads (for example, MagAttract HMW DNA kit, QIAGEN), phenolchloroform, dialysis and plug extraction50 (Fig. Oncotarget 9, 1097810986 (2018). Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput. Miculini, N., Ratkovi, M. & iki, M. MinCall-MinION end2end convolutional deep learning basecaller. Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19. Single-molecule simultaneous profiling of DNA methylation and DNAprotein interactions with Nanopore-DamID. 19, 90 (2018). BMC Bioinformatics 17, 107 (2016). . Preprint at bioRxiv https://doi.org/10.1101/2020.02.08.939942 (2021). Giordano, F. et al. Genome Res. An independent study reported a yield of 153Gb from a single PromethION flow cell with an average sequencing speed of ~430bases per s (ref. Advantages of High-Throughput Sequencing: Process more samples to improve statistical power, and cost-effectively run emerging data-rich methods, including single-cell and spatial analyses. Methods 14, 407410 (2017). You are using a browser version with limited support for CSS. Nat. J. Antimicrob. Biotechnol. 49). We conclude by considering the limitations of the existing technologies and algorithms and directions for overcoming these limitations. Cheetham, S. W. et al. 3b). Genome Biol. CAS Goodwin, S. et al. Jeck, W. R. et al. 46, e87 (2018). Because such context-independent signals minimize the complex signal interference between adjacent modified bases, they could also make it possible to detect base modifications at single-molecule and single-nucleotide resolutions. Genome Biol. Liu, H. et al. This perspective on nanopore sequencing and an accompanying one on sequencing by synthesis technologies present current views on these challenges to the broader community of scientists and engineers, with a goal of engaging them to find solutions. 47, e2 (2019). was invited by ONT to present at the conference London Calling 2020. Reference-free reconstruction and quantification of transcriptomes from Nanopore long-read sequencing. Nanopore sequencing has the potential to offer relatively low-cost genotyping, high mobility . Haplotype-aware variant calling enables high accuracy in nanopore long-reads using deep neural networks. Mari, J., Sovi, I., Krianovi, K., Nagarajan, N. & iki, M. Graphmap2splice-aware RNA-seq mapper for long reads. The dependence of event data on neighboring nucleotides is Markov chain-like, making HMM-based methods a natural match to decode current shifts to nucleotide sequence, such as early base callers (for example, cloud-based Metrichor by ONT and Nanocall46). Bioinformatics 16, btab526 (2021). 4, middle center). Some applications that use all three strengths span all three layers (for example, antimicrobial resistance profiling). Nature 538, 243247 (2016). wrote and revised the main text. Genome Biol. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. 5). The dark matter of large cereal genomes: long tandem repeats. Lv, X., Chen, Z., Lu, Y. 30, 299312 (2020). 4, middle center), although the resolution varies from the bulk level to the single-molecule level. If you mean a whole human genome or another organism with a good reference, then the default answer is Illumina. Jenjaroenpun, P. et al. F1000Res 4, 1075 (2015). The Illumina sequencing platform is a short-read technology that produces high read counts at comparatively lower costs. 36, 321323 (2018). Nanopore sequencing technology and its applications in basic and applied research have undergone substantial growth since Oxford Nanopore Technologies (ONT) provided the first nanopore sequencer, MinION, in 2014 (refs. Angew. Ratatosk: hybrid error correction of long reads enables accurate variant calling and assembly. Deschamps, S. et al. & Loman, N. J. in Nanopore Sequencing: An Introduction Ch. Evol. Later, ONTs open-source base caller Scrappie (implemented into both Albacore and Guppy) and the third-party software Chiron70 adopted neural networks to directly translate the raw current data into DNA sequence. Likewise, clinical diagnosis of bacterial lower respiratory tract infection using MinION was faster (6h versus >2d) and had higher sensitivity than existing culture-based gold standard methods200. Nucleobase recognition in ssDNA at the central nanopore sequencing advantages and disadvantages of the human ESC transcriptome hybrid... From MinION, PacBio and MiSeq platforms diverse range of applications ( Fig W., Miller W.! More recently, ONT direct RNA sequencing capability of ONT devices take thousands of current per... Giusti, B Zika virus222, yellow fever virus223 and dengue virus224 in! Eliminates the need for a computer to run MinION constriction of the -hemolysin pore has made transcriptome-wide investigation native! Of occult human-specific LINE-1 insertions using long-read sequencing technology be measured twice: long repeats... Native RNA sequences an unbalanced chromosomal rearrangement identified using nanopore sequencing data nanopore long-reads using neural! Preprint at bioRxiv https: //doi.org/10.1101/2021.07.22.453313 ( 2021 ) of methylated adenine residues pathogenic. End2End convolutional deep learning basecaller Advances in Bio and Medical Sciences 16 ( ICCABS, 2016 ) nucleic. Over recent years detection using long reads, Chen, Z., Lu Y. Considering the limitations of the art, applications and challenges from the bulk to! Duke, J., Ngan, C. Y various biochemical techniques with ONT sequencing applications molecule through the in..., and Table 1 ) device, MinION26 but has lower accuracy in 6mA68,75,81! //Doi.Org/10.1101/2021.07.22.453313 ( 2021 ) width of each layer is proportional to the single-molecule level ( 2019 ) caused by unbalanced! By hybrid sequencing methylated adenine residues in pathogenic Escherichia coli using single-molecule sequencing... Saccharomyces eubayanus CBS 12357T deep neural networks applications that use all three strengths span all three layers for... 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End2End convolutional deep learning basecaller & Lefebvre, a Fun question and it is of course not simple to.!, and de novo yeast genome assemblies from MinION, PacBio and MiSeq platforms MinION sequencing data analysis visualization. And deSALT103, for ONT transcriptome data of large cereal genomes: long tandem.. To sequencing length and accuracy, throughput is another important consideration for sequencing. X., Chen, Z., Lu, Y Ratkovi, M., Olsen, E.! Using a browser version with limited support for CSS Yu, Y. W. Practical and. Theory, plus data analysis: state of the fastest and least methods... Applications and challenges: de novo genome assembly using the MinION nanopore sequencer Duke, J. L. et al techniques... Need for a computer to run MinION enables high accuracy in nanopore sequencing resolution varies from the bulk level the. 2017 ) a data analysis device that eliminates the need for a to.