Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads

Mueller, Carolin A.; Boemo, Michael A.; Spingardi, Paolo; Kessler, Benedikt M.; Kriaucionis, Skirmantas; Simpson, Jared T.; Nieduszynski, Conrad A.

doi:10.1101/442814

Cited by 32 publications

(66 citation statements)

References 42 publications

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“…Importantly, the nanopore sequencer can detect base modifications on long RNA and DNA molecules [7,8]; for review [9]. In particular, we and others reported the detection of bromodeoxyuridine (BrdU) or other thymidine analogs incorporated during DNA replication in yeast or mouse DNA [10,11,12]. Using S. cerevisiae cells synchronously progressing through S phase in conditions of limiting BrdU concentrations, Müller et al [11] detected gradients of BrdU incorporation along single DNA molecules at 2 kb resolution.…”

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confidence: 99%

“…In particular, we and others reported the detection of bromodeoxyuridine (BrdU) or other thymidine analogs incorporated during DNA replication in yeast or mouse DNA [10,11,12]. Using S. cerevisiae cells synchronously progressing through S phase in conditions of limiting BrdU concentrations, Müller et al [11] detected gradients of BrdU incorporation along single DNA molecules at 2 kb resolution. Importantly, most but not all peaks of BrdU incorporation mapped near known origins, suggesting the existence of dispersed origins too rarely used to be detected by cell population methods.…”

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confidence: 99%

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FORK-seq: replication landscape of theSaccharomyces cerevisiaegenome by nanopore sequencing

Hennion

Arbona

Lacroix

et al. 2020

Preprint

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Genome replication mapping methods profile cell populations, masking cell-tocell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200 nucleotide resolution. By quantifying BrdU incorporation along pulse-chased replication intermediates from Saccharomyces cerevisiae, we orient 58,651 replication tracks reproducing populationbased replication directionality profiles and map 4,964 and 4,485 individual initiation and termination events, respectively. Although most events cluster at known origins and fork merging zones, 9% and 18% of initiation and termination events, respectively, occur at many locations previously missed. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.

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confidence: 99%

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confidence: 99%

FORK-seq: replication landscape of theSaccharomyces cerevisiaegenome by nanopore sequencing

Hennion

Arbona

Lacroix

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…First, better sensitivity and genome coverage could be achieved by incorporating a targeted sequence capture panel [23] although retaining assay simplicity would be a challenge. Second, developing a fully automated sample-to-library procedure would be instrumental for wider use of mNGS and minimization of contaminations in average clinical laboratories.…”

Section: Discussionmentioning

confidence: 99%

A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification

Jia

Wang

et al. 2020

Preprint

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Metagenomic next generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on the assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing based mNGS method was first developed and evaluated using a set of samples with known etiology. Its sensitivity for RNA virus (Influenza A, H1N1) was <6.4×102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then the rapid turnaround time of Nanopore sequencing was testified by sequencing of an Influenza A virus and Adenoviruses. Further, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analyzed and the pathogens identified by illumina sequencing showed 81.8% concordance with qPCR-based results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1 positive patients with meningitis/encephalitis detected HIV-1 RNA and toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol which realized facile metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.

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“…For genome-wide analysis of DNA replication, pulse labeling of replication sites by base analogs, such as BrdU (bromodeoxyuridine), is widely used [71]. Attempts to detect base analogs directly using a Nanopore sequencer have been reported by some groups [72][73][74] (Fig. 3b).…”

Section: Detection Of Dna Replication Using Base Analogsmentioning

confidence: 99%

“…3b). Müllar et al developed D-Nascent (detecting nucleotide analog signal currents on extremely long Nanopore traces) using pulse labeling by BrdU with relatively low toxicity [72]. They applied D-Nascent to analyze replication fork dynamism in S. cerevisiae and achieved the detection of replication sites in reads 20-160 kb in length.…”

Section: Detection Of Dna Replication Using Base Analogsmentioning

confidence: 99%

Recent advances in the detection of base modifications using the Nanopore sequencer

Seki

2019

J Hum Genet

112

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DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.

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Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads

Cited by 32 publications

References 42 publications

FORK-seq: replication landscape of theSaccharomyces cerevisiaegenome by nanopore sequencing

FORK-seq: replication landscape of theSaccharomyces cerevisiaegenome by nanopore sequencing

A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification

Recent advances in the detection of base modifications using the Nanopore sequencer

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