Nanopore sequencing and assembly of a human genome with ultra-long reads

Jain, Miten; Koren, Sergey; Miga, Karen H.; Quick, Josh; Rand, Arthur C; Sasani, Thomas A; Tyson, John R.; Beggs, Andrew D; Dilthey, Alexander; Fiddes, Ian T.; Malla, Sunir; Marriott, Hannah; Nieto, Tom; O’Grady, Justin; Olsen, Hugh E.; Pedersen, Brent S.; Rhie, Arang; Richardson, Hollian; Quinlan, Aaron R.; Snutch, Terrance P.; Tee, Louise; Paten, Benedict; Phillippy, Adam M.; Simpson, Jared T.; Loman, Nicholas J.; Loose, Matthew

doi:10.1038/nbt.4060

Cited by 1,652 publications

(1,682 citation statements)

References 66 publications

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“…We observed that the errors tend to occur between purines or between pyrimidines, probably because they have similar chemical structures 11 . In addition, we also observed that substitution errors tend to occur at the same nucleotide position across repeats (Supplemental Fig.…”

Section: Discussionmentioning

confidence: 84%

“…The advantage of the nanopore sequencer over the PacBio sequencer is the ultra-long read capability 11 . It has the potential to obtain reads of more than 100 kbp, the approximate mean size of D4Z4 in healthy individuals.…”

Section: Discussionmentioning

confidence: 99%

“…It has the potential to obtain reads of more than 100 kbp, the approximate mean size of D4Z4 in healthy individuals. Currently, we could only obtain two reads that potentially cover all chr4 D4Z4 repeats from human genome data with 5 × coverage using 14 flow-cells 11 . In that paper, they were not able to complete an alignment of the ultra-long reads using BWA-MEM.…”

Section: Discussionmentioning

confidence: 99%

“…Sequencing of this D4Z4 array using Sanger sequencing or short-read sequencers (up to 300 bp for Illumina and IonTorrent) is technically difficult owing to the high similarity and the high GC content of the repeats. The Oxford Nanopore Technologies MinION (Oxford, UK) is a single-molecule sequencer that can produce long reads exceeding 100 kbp 11 . Therefore, MinION sequencing may enable the determination of pathogenicity by sequencing the complete D4Z4 array.…”

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confidence: 99%

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Nanopore-based single molecule sequencing for D4Z4 array responsible for facioscapulohumeral muscular dystrophy

Mitsuhashi

Ueda

et al. 2017

Journal of the Neurological Sciences

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Subtelomeric macrosatellite repeats are difficult to sequence using conventional sequencing methods owing to the high similarity among repeat units and high GC content. Sequencing these repetitive regions is challenging, even with recent improvements in sequencing technologies. Among these repeats, a haplotype carrying a particular sequence and shortening of the D4Z4 array on human chromosome 4q35 causes one of the most prevalent forms of muscular dystrophy with autosomaldominant inheritance, facioscapulohumeral muscular dystrophy (FSHD). Here, we applied a nanoporebased ultra-long read sequencer to sequence a BAC clone containing 13 D4Z4 repeats and flanking regions. We successfully obtained the whole D4Z4 repeat sequence, including the pathogenic gene DUX4 in the last D4Z4 repeat. The estimated sequence accuracy of the total repeat region was 99.8% based on a comparison with the reference sequence. Errors were typically observed between purine or between pyrimidine bases. Further, we analyzed the D4Z4 sequence from publicly available ultra-long whole human genome sequencing data obtained by nanopore sequencing. This technology may be a new tool for studying D4Z4 repeats and pathomechanism of FSHD in the future and has the potential to widen our understanding of subtelomeric regions.Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent adult-onset muscular dystrophies. The genomes of most patients with FSHD have a common feature, i.e., a contracted subtelomeric macrosatellite repeat array called D4Z4 on chromosome 4q35. The D4Z4 array consists of a highly similar 3.3-kb single repeat unit. Normally, the D4Z4 array is highly methylated and forms heterochromatin. Patients with FSHD have less than 11 D4Z4 repeats 1-3 . In Japan, the majority of patients with FSHD have less than 7 repeats 4 . Shortening of the D4Z4 array causes the de-repression of the flanking genes as well as DUX4, located in the last D4Z4 repeat. The ectopic expression of DUX4 is toxic in muscle tissues and is thought to be a causal factor for FSHD [5][6][7][8][9] . In addition to the repeat number, the haplotype of the last D4Z4 repeat is important for the development of FSHD 1,2 . The telomeric flanking region of D4Z4 contains the 3′ UTR of DUX4 and is called the pLAM region. The presence of a polyadenylation signal in this region allows DUX4 expression and disease manifestation 10 . In contrast, individuals without polyadenylation signals do not manifest the disease 2 .

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Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Nanopore-based single molecule sequencing for D4Z4 array responsible for facioscapulohumeral muscular dystrophy

Mitsuhashi

Ueda

et al. 2017

Journal of the Neurological Sciences

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“…An alternative approach may be to use nanopore sequencers to directly read different modifications from the DNA (Burgess 2017; Schatz 2017). Nanopore sequencing uses pores through which nucleic acid strands are Bpulled^and the ionic pattern reveals the nucleotide sequence, including modifications (Jain et al 2017;Shendure et al 2017). These technologies are still in the developmental phase but show great promise (Rand et al 2017).…”

Section: Alternatives To Conversion-based Sequencingmentioning

confidence: 99%

Analysis of DNA modifications in aging research

et al. 2018

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As geroscience research extends into the role of epigenetics in aging and age-related disease, researchers are being confronted with unfamiliar molecular techniques and data analysis methods that can be difficult to integrate into their work. In this review, we focus on the analysis of DNA modifications, namely cytosine methylation and hydroxymethylation, through nextgeneration sequencing methods. While older techniques for modification analysis performed relative quantitation across regions of the genome or examined average genome levels, these analyses lack the desired specificity, rigor, and genomic coverage to firmly establish the nature of genomic methylation patterns and their response to aging. With recent methodological advances, such as whole genome bisulfite sequencing (WGBS), bisulfite oligonucleotide capture sequencing (BOCS), and bisulfite amplicon sequencing (BSAS), cytosine modifications can now be readily analyzed with base-specific, absolute quantitation at both cytosine-guanine dinucleotide (CG) and non-CG sites throughout the genome or within specific regions of interest by next-generation sequencing. Additional advances, such as oxidative bisulfite conversion to differentiate methylation from hydroxymethylation and analysis of limited input/single-cells, have great promise for continuing to expand epigenomic capabilities. This review provides a background on DNA modifications, the current state-of-theart for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.

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Genetic basis of mitochondrial diseases

Gusic

2021

FEBS Letters

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Mitochondrial disorders are monogenic disorders characterized by a defect in oxidative phosphorylation and caused by pathogenic variants in one of over 340 different genes. The implementation of whole exome sequencing has led to a revolution in their diagnosis, duplicated the number of associated disease genes, and significantly increased the diagnosed fraction. However, the genetic etiology of a substantial fraction of patients exhibiting mitochondrial disorders remains unknown, highlighting limitations in variant detection and interpretation, which calls for improved computational and DNA sequencing methods, as well as the addition of OMICS tools. More intriguingly, this also suggests that some pathogenic variants lie outside of the proteincoding genes and that the mechanisms beyond the Mendelian inheritance and the mtDNA are of relevance. This review covers the current status of the genetic basis of mitochondrial diseases, discusses current challenges and perspectives, and explores the contribution of factors beyond the protein-coding regions and monogenic inheritance in the expansion of the genetic spectrum of disease.

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Nanopore sequencing and assembly of a human genome with ultra-long reads

Cited by 1,652 publications

References 66 publications

Nanopore-based single molecule sequencing for D4Z4 array responsible for facioscapulohumeral muscular dystrophy

Nanopore-based single molecule sequencing for D4Z4 array responsible for facioscapulohumeral muscular dystrophy

Analysis of DNA modifications in aging research

Genetic basis of mitochondrial diseases

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