Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

Ardui, Simon; Ameur, Adam; Vermeesch, Joris Robert; Hestand, Matthew S.

doi:10.1093/nar/gky066

Cited by 569 publications

(397 citation statements)

References 98 publications

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“…Long-read sequencers, such as PacBio R SMRT II and Oxford Nanopore MinION, can provide species-and strain-level microbiota resolution, but their low throughput increases their error rates, which makes hybrid sequencing the method of choice for comprehensive microbiome analysis. 70,71 Hybrid sequencing, involving the combination of long-and short-read sequencing, has been shown to produce better metagenomic read lengths and coverage/depth than using single sequencing platforms. 71,72 Although expensive, hybrid sequencing yields better metagenomic and transcriptomic sequence reads for a better resolution of the sputum/BAL and gut microbiome species, as well as gene expression profiles to elucidate the interactions between Mtb, the immune system, and commensals.…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

Mycobacterium tuberculosis, antimicrobials, immunity, and lung–gut microbiota crosstalk: current updates and emerging advances

Sekyere

Maningi

Fourie

2020

Annals of the New York Academy of Sciences

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Increasingly, gut microbiota distortions are being implicated in the pathogenesis of several infectious and noninfectious diseases. Specifically, in the absence of an eubiotic microbiota, mice are more prone to colonization and infection by Mycobacterium tuberculosis (Mtb). In this qualitative analysis, the following were observed: (1) antimicrobials cause long-term gut microbiota perturbations; (2) Mtb causes limited and transient disturbances to the lung-gut microbiota; (3) pathogens (e.g., Helicobacter hepaticus) affect microbiota integrity and reduce resistance to Mtb; (4) dysbiosis depletes bacterial species regulating proper immune functioning, reducing resistance to Mtb; (5) dysregulated immune cells fail to express important pathogen-recognition receptors (e.g., macrophageinducible C-type lectin; MINCLE) and Mtb-killing cytokines (e.g., IFN-γ, TNF-α, and IL-17), with hampered phagocytic capability; (6) autophagy is central to the immune system's clearance of Mtb, control of inflammation, and immunity-microbiome balance; (7) microbiota-produced short-chain fatty acids, which are reduced by dysbiosis, affect immune cells and increase Mtb proliferation; (8) commensal species (e.g., Lactobacillus plantarum) and microbiota metabolites (e.g., indole propionic acid) reduce tuberculosis progression; and (9) fecal transplants mostly restored eubiosis, increased immune resistance to Mtb, restricted dissemination of Mtb, and reduced tuberculosis-associated organ pathologies. Overuse of antimicrobials, as shown in mice, is a risk factor for reactivating latent or treated tuberculosis.

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Section: Challenges and Future Perspectivesmentioning

confidence: 99%

Mycobacterium tuberculosis, antimicrobials, immunity, and lung–gut microbiota crosstalk: current updates and emerging advances

Sekyere

Maningi

Fourie

2020

Annals of the New York Academy of Sciences

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“…In order to analyse amplicons larger than 2000 bp in high throughput, the only feasible approach would be to use long-read sequencing technologies such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio). However, these methods are limited by a relatively high raw error-rate of 9 and 13% respectively 17 . For PacBio, the circular consensus sequencing (CCS) approach, where a template molecule is circularized and read multiple times by the polymerase, can produce mean error rates to as low as 0.04% 18 .…”

Section: Introductionmentioning

confidence: 99%

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Karst

Ziels

Kirkegaard

et al. 2019

Preprint

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High-throughput amplicon sequencing of large genomic regions represents a challenge for existing short-read technologies. Long-read technologies can in theory sequence large genomic regions, but they currently suffer from high error rates. Here, we report a highthroughput amplicon sequencing approach that combines unique molecular identifiers (UMIs) with Oxford Nanopore sequencing to generate single-molecule consensus sequences of large genomic regions. We demonstrate the approach by generating nearly 10,000 full-length ribosomal RNA (rRNA) operons of roughly 4,400 bp in length from a mock microbial community consisting of eight bacterial species using a single Oxford Nanopore MinION flowcell. The mean error rate of the consensus sequences was 0.03%, with no detectable chimeras due to a rigorous UMI-barcode filtering strategy. The simplicity and accessibility of this method paves way for widespread use of high-accuracy amplicon sequencing in a variety of genomic applications.

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“…SMRT sequencing is performed by continuously observing the incorporation of a fluorescently labeled nucleotide by DNA polymerase in each well. Approximately 365,000 reads with an average length of 10-14 kb are generated by the PacBio Sequel sequencer per an SMRT cell [42]. On the basis of differences in the duration of nucleotide incorporation between unmodified and modified bases, SMRT sequencing can detect the positions and kinds of base modifications (Fig.…”

Section: Smrt (Single Molecule Real-time) Sequencingmentioning

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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Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

Cited by 569 publications

References 98 publications

Mycobacterium tuberculosis, antimicrobials, immunity, and lung–gut microbiota crosstalk: current updates and emerging advances

Mycobacterium tuberculosis, antimicrobials, immunity, and lung–gut microbiota crosstalk: current updates and emerging advances

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

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

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