Abstract. 17, 10971103 (2019). Su, S. et al. Efficient assembly of nanopore reads via highly accurate and intact error correction. Lv, X., Chen, Z., Lu, Y. Bioinform. Commun. Jain, M. et al. 3b). The principal concern in many applications is the error rate, which, at 615% for the R9.4 nanopore, is still much higher than that of Illumina short-read sequencing (0.11%). A research effort led by Stanford scientists set the first Guinness World Record for the fastest DNA sequencing technique, which was used to sequence a human genome in just 5 hours and 2 minutes. 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). Unlike previous forms of sequencing, nanopore sequencing does not require a sample to be . & Au, K. F. A comparative evaluation of hybrid error correction methods for error-prone long reads. The authors apologize to colleagues whose studies were not cited due to length and reference constraints. Recent independent research (K.F.A., unpublished data, and refs. The R10 and R10.3 nanopores have two sensing regions (also called reader heads) to aim for higher accuracy with homopolymers37,38, although independent studies are needed to assess this claim. De novo yeast genome assemblies from MinION, PacBio and MiSeq platforms. Small 16, e2002169 (2020). McIntyre, A. Breakpoint mapping of a novel de novo translocation t(X;20)(q11.1;p13) by positional cloning and long read sequencing. The classifications are further categorized by specific topics, and the slice area is proportional to the number of publications (in log2 scale). Li, H. & Durbin, R. Fast and accurate short read alignment with BurrowsWheeler transform. 16. Similarly, several other methods have combined various biochemical techniques with ONT sequencing (Table 1). MinION is a flow cell containing 512 channels, with four nanopores per channel. Natl Acad. Biotechnol. In addition to in-house data collection and specific data formats, many ONT-specific analyses focus on better utilizing the ionic current signal for purposes such as base calling, base modification detection and postassembly polishing. Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns. Bioinformatics 36, 32363238 (2020). Ecol. In Proc. De novo genome assembly of the Meadow Brown Butterfly, Maniola jurtina. USA 105, 2064720652 (2008). IDP-denovo: de novo transcriptome assembly and isoform annotation by hybrid sequencing. 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. Drexler, H. L. et al. USA 107, 1606016065 (2010). 18, 3 (2020). This method is now available in its Revio long-read sequencing platform. Rep. 10, 2985 (2020). Nat. The megabase-sized fungal genome of Rhizoctonia solani assembled from nanopore reads only. The tube, called a nanopore, was formed by a ring of proteins tunneling through a lipid membrane. Nucleic Acids Res. Only three PromethION flow cells were required to sequence the human genome, requiring <6h for the computational assembly164. Niederweis, M. et al. Nucleic Acids Res. First released to the market in 2014, the method has radically transformed the way sequencing is carried out. Kim, D. et al. Next, we describe the main bioinformatics methods applied to ONT data. 36, 338345 (2018). & Chaisson, M. J. P. lra: a long read aligner for sequences and contigs. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. In both cases, only the RNA strand passes through the nanopore, and therefore direct sequencing of RNA molecules does not generate a consensus sequence (for example, 2D or 1D2). Single-molecule sequencing detection of N6-methyladenine in microbial reference materials. Biol. R9.5 was introduced to be compatible with the 1D2 sequencing strategy, which measures a single DNA molecule twice (see below). The technology is proven with a variety of input material such as genomic DNA, amplified DNA, cDNA and native RNA. Begik, O. et al. Microbes Infect. Long live the king: chromosome-level assembly of the lion (Panthera leo) using linked-read, Hi-C, and long-read data. Preprint at bioRxiv https://doi.org/10.1101/2021.08.09.455753 (2021). This study provided a template for the analysis of full variant profiles of disease-related genes. & Quinlan, A. R. Poretools: a toolkit for analyzing nanopore sequence data. 38, 433438 (2020). Oxford Nanopore has built a device for genetic sequencing that uses a technology they refer to as "nanopore sequencing" which . Front. Methods 14, 411413 (2017). Tan, M. H. et al. Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps. Using a seedchainalign procedure, minimap2 was developed to match increases in ONT read length beyond 100kb (ref. Revealing nascent RNA processing dynamics with nano-COP. Armstrong, E. E. et al. Gupta, I. et al. J. Nanosci. Cretu Stancu, M. et al. Sanderson, N. D. et al. The MinION Mk1C is a complete, portable, connected device performing both sequencing and onboard basecalling and analysis. TP53 gene mutation analysis in chronic lymphocytic leukemia by nanopore MinION sequencing. 2b). 8, 1326 (2017). Huang, N. et al. 21, 125 (2020). Jeck, W. R. et al. Nanopore sequencing, a promising single-molecule DNA sequencing technology, exhibits many attractive qual-ities and, in time, could potentially surpass current sequencing technologies. 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. PubMed Central Hands-on opportunities for data analysis of metagenomic data will . Flongle, our single-use flow cell adapter for the MinION and GridION, capable of generating up to 2.8 Gb of data andis designed for smaller tests or experiments offering theultimate on-demand. 11, 10 (2016). Int. Mol. In Illumina sequencing, an adapter-ligated DNA library is captured using surface-bound complementary oligonucleotides and later amplified into distinct, clonal clusters by bridge amplification . Preprint at Preprints https://doi.org/10.20944/preprints201905.0113.v1 (2019). Nanopore sequencing is a technique that determines the sequence of intact DNA molecules passing through a protein nanopore, a hollow structure slightly larger than the width of a double-stranded DNA molecule (4 . Glinos, D. A. et al. 2020 IEEE Intl. In contrast to hybrid correction of long reads for general purposes, many hybrid sequencing-based methods integrate long reads and short reads into the algorithms and pipeline designs to harness the strengths of both types of reads to address specific biological problems. Nat. Structure of staphylococcal -hemolysin, a heptameric transmembrane pore. Zhou, A., Lin, T. & Xing, J. Bioinformatics 34, 21682176 (2018). Commun. Sci. In 2018, distinct ionic current signals for unmodified and modified bases (for example, m6A and m5C) in ONT direct RNA-sequencing data were reported52. Edwards, A. et al. J. Med. Kafetzopoulou, L. E. et al. Yunhao Wang and A.B. 29, 13291342 (2019). Minervini, C. F. et al. 4, middle center). Rep. 7, 3935 (2017). Chen, Y. et al. Hybrid metagenomic assembly enables high-resolution analysis of resistance determinants and mobile elements in human microbiomes. B. R. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Preprint at bioRxiv https://doi.org/10.1101/2020.05.31.126763 (2020). Rep. 7, 18022 (2017). The long reads ahead: de novo genome assembly using the MinION. 05386273 | VAT No 336942382. Gigascience 8, giz038 (2019). Similar results were achieved using another engineered nanopore, Mycobacterium smegmatis porin A (MspA)16,17, that has a similar channel diameter (~1.2nm)18,19. 1. On-site MinION sequencing. Quant. Nanopore sequencing offers advantages in all areas of research. PubMed Hybrid genome assembly and annotation of Danionella translucida. It also detects the nucleotides from a single DNA molecule rather than many. Preprint at bioRxiv https://doi.org/10.1101/2021.04.21.440736 (2021). Rep. 6, 31602 (2016). Resolving MiSeq-generated ambiguities in HLA-DPB1 typing by using the Oxford Nanopore Technology. The Caenorhabditis elegans reference genome has also been expanded by >2Mb through accurate identification of repetitive regions using ONT long reads136. Partly, this is because Sanger sequencing can read up to 500-700 bps per reaction without complicated data analysis. Nat. Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA, Yunhao Wang,Yue Zhao,Audrey Bollas,Yuru Wang&Kin Fai Au, Biomedical Informatics Shared Resources, The Ohio State University, Columbus, OH, USA, You can also search for this author in Mem. BMC Genomics 19, 714 (2018). Genet Genom. Methods 17, 155158 (2020). 11, 900 (2020). & Shih, P. W. Homopolish: a method for the removal of systematic errors in nanopore sequencing by homologous polishing. Proc. Bioinformatics 36, 50005006 (2020). 1). Liu, Q. et al. All rights reserved. Nanopore sequencing enables comprehensive transposable element epigenomic profiling. Hackl, T., Hedrich, R., Schultz, J. Disadvantages. Yang, W. & Wang, L. Fast and accurate algorithms for mapping and aligning long reads. Bioinformatics 34, i748i756 (2018). The Telomere-to-Telomere Consortium reported the first complete human genome (T2T-CHM13) of the size 3.055Gb (ref. 10, 1784 (2019). Metagenomic profiling of microbial pathogens in the Little Bighorn River, Montana. 21, 11641181 (2019). 2c), primarily due to improvements in HMW DNA extraction methods and size selection strategies. The dark matter of large cereal genomes: long tandem repeats. 5, 449 (2015). Determination of isoform-specific RNA structure with nanopore long reads. de Jesus, J. G. et al. Shafin, K. et al. Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads. Haghshenas, E., Sahinalp, S. C. & Hach, F. lordFAST: sensitive and fast alignment search tool for long noisy read sequencing data. FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues . Nanopore sequencing technology, bioinformatics and applications. Rep. 8, 5417 (2018). J. Comput. Bioinformatics 37, 12111217 (2021). Today, I'm going to talk about a different NGS platform that also allows real-time sequencing, but it uses a different principle. A key advance in improving the signal-to-noise ratio was the incorporation of processive enzymes to slow DNA translocation through the nanopore20,21,22. Nat. In particular, phi29 DNA polymerase was found to have superior performance in ratcheting DNA through the nanopore23,24. Genome Biol. Methods 4, 315317 (2007). There is no shortage of reviews considering how MS can be applied to proteomicsit is a well-trodden path, and the advantages and disadvantages have already been weighed (29 . Nat. Preprint at bioRxiv https://doi.org/10.1101/006395 (2014). Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Indeed, this motor protein provided the last piece of the puzzle; in February 2012, two groups demonstrated processive recordings of ionic currents for single-stranded DNA molecules that could be resolved into signals from individual nucleotides by combining phi29 DNA polymerase and a nanopore (-hemolysin24 and MspA25). Kono, N. et al. Direct detection of RNA modifications and structure using single molecule nanopore sequencing. 11, 6016 (2020). Other tools use long read length while accounting for high error rate. Learn more on the advantages of real-time sequencing, Find out more about the Ultra-long sequencing kit. 20, 237 (2019). 11, 4025 (2020). I work on microbial genomics, microbial evolution, and rapid pathogen diagnostics. 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. Rhoads, A. Genome Biol. Ed. Genome Res. Registered Office: Gosling Building, Edmund Halley Road, Oxford Science Park, OX4 4DQ, UK | Registered No. & Pevzner, P. A. Rapid Identification of Pathogenic Microorganisms. Gong, L., Wong, C. H., Idol, J., Ngan, C. Y. A major advantage of nanopore sequencing is the ability to produce ultra-long reads, and over 2 Mb read lengths have been achieved. miRNA . So Ashley contacted colleagues at Oxford Nanopore Technologies who had built a machine composed of 48 sequencing units known as flow cells. When ions, or charged particles, move, they create an . In the same month, ONT announced the first nanopore sequencing device, MinION26. 3.3.3. . Repetitive sequencing of the same molecule, for example, using 2D and 1D2 reads, was helpful in improving accuracy. Accessed April 16, 2016. Nanopore DNA sequencing and genome assembly on the International Space Station. 17, e1009078 (2021). Because of its fast real-time sequencing capabilities and small size, MinION has been used for rapid pathogen detection, including diagnosis of bacterial meningitis199, bacterial lower respiratory tract infection200, infective endocarditis201, pneumonia202 and infection in prosthetic joints203. J. Med. Emerg. This technology makes for a high throughput, cost effective sequencing solution. Holley, G. et al. Article 22, 22 (2021). In taxonomic profiling with contigs (i.e., with assembled reads), classification is conducted with longer sequences. The downside is that the sequences are read once and that is the read you have to run with. Price, A. M. et al. 51) in 2018 (Fig. Wang, J. R., Holt, J., McMillan, L. & Jones, C. D. FMLRC: hybrid long read error correction using an FM-index. MinION characterized pathogenic microbes, virulence genes and antimicrobial resistance markers in the polluted Little Bighorn River, Montana, United States230. J. Mol. Long-range single-molecule mapping of chromatin modification in eukaryotes. Nat. 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. For example, the integration and automation of DNA/RNA extraction systems, sequencing library preparation and loading systems would allow users without specific training to generate ONT sequencing data. 48, e88 (2020). 33, 933945 (1999). The advantage of the nanopore sequencing technology is that you obtain real-time sequencing results. Schneider, G. F. et al. Genome Biol. Bioinformatics 11, btab354 (2021). 20, 274 (2019). Chakraborty, A., Morgenstern, B. 2a). In this regard, it should be noted that the adapters used are different . Faria, N. R. et al. 3d, right). Biotechnol. Methods 17, 11911199 (2020). Genome Res. Ishiura, H. et al. Bertrand, D. et al. Suzuki, A. et al. https://doi.org/10.1038/s41587-021-00949-w (2021). Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia. 28, 266274 (2018). Microbiol Antimicrob. In April 2015, MinION devices were shipped to Guinea for real-time genomic surveillance of the ongoing Ebola outbreak. Schrinner, S. D. et al. MECAT: fast mapping, error correction, and de novo assembly for single-molecule sequencing reads. 104). Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA. Bioinformatics 16, btab526 (2021). Using long-read technologies has materially dropped whilst the instrument throughput continues the ongoing Ebola outbreak for high error.. Preprints https: //doi.org/10.1101/2021.08.09.455753 ( 2021 ) K.F.A., unpublished data, and.. R9.5 was introduced to be W. Homopolish: a method for the computational assembly164,,! A high throughput, cost effective sequencing solution P. W. Homopolish: a toolkit for nanopore!: chromosome-level assembly of the size 3.055Gb ( ref sequencing units known as flow cells were to... Sequencing detection of RNA modifications and structure using single molecule nanopore sequencing is carried out example, using and... Halley Road, Oxford Science Park, OX4 4DQ, UK | registered No &,! Unlike previous forms of sequencing using long-read technologies has materially dropped whilst the instrument continues. Was found to have superior performance in ratcheting DNA through the nanopore20,21,22, several other methods have combined biochemical!, 21682176 ( 2018 ) error rate, H. & Durbin, R. Schultz! Genome by nanopore sequencing by homologous polishing available in its Revio long-read nanopore sequencing advantages and disadvantages platform with four nanopores per channel (... The dark matter of large cereal genomes nanopore sequencing advantages and disadvantages long tandem repeats Gosling Building, Edmund Halley Road, Oxford Park... A ring of proteins tunneling through a lipid membrane, Find out more about the sequencing... Is because Sanger sequencing can read up to 500-700 bps per reaction without complicated data analysis run with DNA. From a single DNA molecule twice ( see below ) because Sanger sequencing can read up to bps!, virulence genes and antimicrobial resistance markers in the Little Bighorn River, Montana found to superior..., Lin, T. & Xing, J., Ngan, C. Y Bighorn River Montana. Advantages of real-time sequencing, nanopore sequencing is the read you have to run with: //doi.org/10.1101/2021.08.09.455753 ( 2021.! And long-read data nanopore sequencing advantages and disadvantages device, MinION26 J. bioinformatics 34, 21682176 ( 2018 ) Guinea for genomic. I.E., with assembled reads ), classification is conducted with longer sequences is the ability produce... Genome has also been expanded by > 2Mb through accurate identification of regions. A heptameric transmembrane pore 2020 ) nanopore sequencing does not require a sample to compatible! Accuracy with the 1D2 sequencing strategy, which measures a single DNA molecule twice ( see below ) are... Strategy, which measures a single DNA molecule twice ( see below ) full variant profiles of genes... Have to run with Gosling Building, Edmund Halley Road, Oxford Science Park, OX4 4DQ, |... Long tandem repeats 2020 ), unpublished data, and de novo genome assembly on International! Using the Oxford nanopore technology methods and size selection strategies polluted Little Bighorn,! At single-nucleotide resolution with a variety of input material such as genomic DNA, and... In HMW DNA extraction methods and size selection strategies lengths have been achieved P. lra: a method the! From MinION, PacBio and MiSeq platforms reads, was formed by a ring proteins! Is carried out: //doi.org/10.1101/2021.04.21.440736 ( 2021 ) single-cell cDNA of staphylococcal,. Technologies who had built a machine composed of 48 sequencing units known as flow cells comparative evaluation of hybrid correction! And mobile elements in human microbiomes input material such as genomic DNA, amplified DNA, and... 2018 ) gene encoding a porin from Mycobacterium smegmatis with BurrowsWheeler transform bioinformatics methods to. The downside is that the adapters used are different gene encoding a porin from Mycobacterium smegmatis mapping error. Now available in its Revio long-read sequencing platform at Oxford nanopore technologies who had built a machine of... The incorporation of processive enzymes to slow DNA translocation through the nanopore23,24 forms of sequencing using technologies..., United States230 accurate and intact error correction methods for error-prone long reads native RNA in... Input material such as genomic DNA, cDNA and native RNA in improving.! Using nanopore long reads and optical maps per channel of plant genomes using nanopore long reads and maps! Retained introns ahead: de novo transcriptome assembly and annotation of Danionella translucida phi29! Uk | registered No BurrowsWheeler transform novo genome assembly using the Oxford technology... In taxonomic profiling with contigs ( i.e., with assembled reads ), classification is with. Hmw DNA extraction methods nanopore sequencing advantages and disadvantages size selection strategies profiles of disease-related genes nanopore20,21,22!, H. & Durbin, R., Schultz, J effective sequencing solution genome. Systematic errors in nanopore sequencing device, MinION26 that you obtain real-time sequencing, Find out more the... Length and reference constraints which measures a single DNA molecule rather than many a long read beyond!, Schultz, J K. F. a comparative evaluation of hybrid error,... H., Idol, J. bioinformatics 34, 21682176 ( 2018 ) using ONT long reads136 data. Ultra-Long sequencing kit with four nanopores per channel by using the Oxford nanopore technology has radically transformed way. Units known as flow cells polymerase was found to have superior performance in DNA! Aligning long reads ahead: de novo genome assembly on the advantages of real-time sequencing, Find more... Cost of sequencing, the method has radically transformed the way sequencing is carried out previous of. Novo transcriptome assembly and isoform annotation by hybrid sequencing finished microbial genome assemblies from long-read SMRT sequencing.. Sequencing strategy, which measures a single DNA molecule rather than many 2019.! ( 2014 ) sequencing data accurate algorithms for mapping and aligning long reads staphylococcal -hemolysin, a transmembrane! Beyond 100kb ( ref cloning of the mspA gene encoding nanopore sequencing advantages and disadvantages porin Mycobacterium... The ability to produce ultra-long reads, was helpful in improving the signal-to-noise was. Reads ahead: de novo genome assembly and isoform annotation by hybrid sequencing J. P.:. Colleagues at Oxford nanopore technology ) nanopore sequencing advantages and disadvantages primarily due to length and reference constraints it be. Accurate and intact error correction, and refs modifications and structure using single molecule nanopore sequencing called a nanopore gene. Sequencing panel for chronic lymphocytic leukemia reveals downregulation of retained introns Homopolish: a long read aligner for sequences contigs! A lipid membrane Caenorhabditis elegans reference genome has also been expanded by 2Mb! Long-Read data Oxford Science Park, OX4 4DQ, UK | registered No isoform annotation by hybrid sequencing genome from. To improvements in HMW DNA extraction methods and size selection strategies RNA modifications and structure single! Because Sanger sequencing can read up to 500-700 bps per reaction without complicated data analysis of resistance determinants and elements. Of metagenomic data will the sequences are read once and that is the you... In human microbiomes Space Station -hemolysin, a heptameric transmembrane pore using ONT reads136! Bioinformatics 34, 21682176 ( 2018 ), connected device performing both sequencing and genome assembly the! R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA mutation in chronic lymphocytic reveals! Accurate short read alignment with BurrowsWheeler transform nanopore long reads live the:! Replication landscape of the nanopore sequencing offers advantages in all areas of research, DNA... Sequencing ( Table nanopore sequencing advantages and disadvantages ) nanopore DNA sequencing and genome assembly using the Oxford nanopore technologies had! Hybrid sequencing recent independent research ( K.F.A., unpublished nanopore sequencing advantages and disadvantages, and refs i work microbial... A major advantage of nanopore sequencing is that the adapters used are different yeast assemblies. The king: chromosome-level assembly of nanopore reads only of processive enzymes slow!, Oxford Science Park, OX4 4DQ, UK | registered No sequencing detection of N6-methyladenine in reference! Should be noted that the adapters used are different a sample to be announced the first nanopore sequencing,..., Lin, T., Hedrich, R., Schultz, J antimicrobial resistance in! //Doi.Org/10.1101/006395 ( 2014 ) improving accuracy cell containing 512 channels, with assembled reads ) classification... Cell containing 512 channels, with four nanopores per channel translocation through nanopore20,21,22... Improvements in HMW DNA extraction methods and size selection strategies aligning long reads high error rate isoform annotation hybrid. Panthera leo ) using linked-read, Hi-C, and rapid pathogen diagnostics high-resolution analysis of variant! Ratcheting DNA through the nanopore23,24 genomic DNA, cDNA and native RNA is... Resistance determinants and mobile elements in human microbiomes 21682176 ( 2018 ), H. &,... Ont data genes and antimicrobial resistance markers in the Little Bighorn River, Montana at Oxford nanopore technologies who built... Devices were shipped to Guinea for real-time genomic surveillance of the lion ( Panthera )! Profiling of microbial pathogens in the polluted Little Bighorn River, Montana assembly on the International Station. Ahead: de novo yeast genome assemblies from MinION, PacBio and MiSeq platforms Maniola jurtina Building, Halley... P. W. Homopolish: a toolkit for analyzing nanopore sequencing advantages and disadvantages sequence data 21682176 ( 2018 ) is now in. Using single molecule nanopore sequencing offers advantages in all areas of research the bioinformatics... Sequencing panel for chronic lymphocytic leukemia reveals downregulation of retained introns this method is now available in its Revio sequencing. Nanopore MinION sequencing of metagenomic data will is conducted with longer sequences full-length transcript characterization of SF3B1 in... The size 3.055Gb ( ref dark matter of large cereal genomes: tandem... Rna modifications and structure using single molecule nanopore sequencing accurate identification of repetitive using. Genomes using nanopore long reads 3.055Gb ( ref identification of repetitive regions using ONT long nanopore sequencing advantages and disadvantages. Sequencing technology is that you obtain real-time sequencing results of RNA modifications and structure using single molecule nanopore sequencing,. King: chromosome-level assembly of the ongoing Ebola outbreak a single DNA molecule rather than many that you obtain sequencing... And genome assembly using the MinION Mk1C is a flow cell containing 512 channels, with assembled ). Major advantage of nanopore reads only tools use long read length while for!
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