A world of opportunities with nanopore sequencing

Leggett, Richard M.; Clark, Matthew D.

doi:10.7287/peerj.preprints.3090v1

Cited by 27 publications

(40 citation statements)

References 29 publications

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“…18,19 Despite its limited throughput and moderate sequencing accuracy to date, several outstanding features such as its portability, read out of ultra-long reads (>100 kb), and the immediate processing of generated reads in real time make this technology highly promising for many different applications. 20 Of interest, the potential of this sequencing platform with respect to clinical microbiology has already been shown for several studies ranging from outbreak surveillances to infection diagnostics from various clinical samples (urine, blood, implants, feces, sputum) and antimicrobial resistance profiling. 21e27 However, for sepsis diagnostics of patient samples, such a rapid real-time sequencing-based workflow has not been established yet.…”

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confidence: 99%

Rapid Next-Generation Sequencing–Based Diagnostics of Bacteremia in Septic Patients

Grumaz

Hoffmann

Vainshtein

et al. 2020

The Journal of Molecular Diagnostics

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The increasing incidence of bloodstream infections including sepsis is a major challenge in intensive care units worldwide. However, current diagnostics for pathogen identification mainly depend on culture-and molecular-based approaches, which are not satisfactory regarding specificity, sensitivity, and time to diagnosis. Herein, we established a complete diagnostic workflow for real-time highthroughput sequencing of cell-free DNA from plasma based on nanopore sequencing for the detection of the causative agents, which was applied to the analyses of eight samples from four septic patients and three healthy controls, and subsequently validated against standard next-generation sequencing results. By optimization of library preparation protocols for short fragments with low input amounts, a 3.5-fold increase in sequencing throughput could be achieved. With tailored bioinformatics workflows, all eight septic patient samples were found to be positive for relevant pathogens. When considering time to diagnosis, pathogens were identified within minutes after start of sequencing. Moreover, an extrapolation of real-time sequencing performance on a cohort of 239 septic patient samples revealed that more than 90% of pathogen hits would have also been detected using the optimized MinION workflow. Reliable identification of pathogens based on circulating cell-free DNA sequencing using optimized workflows and real-time nanopore-based sequencing can be accomplished within 5 to 6 hours following blood draw. Therefore, this approach might provide therapy-relevant results in a clinically critical timeframe. (J Mol Diagn 2020, 22: 405e418; https://doi.Bloodstream infections, in particular sepsis, represent one of the main causes of death, with a mortality rate of up to 30% to 50% in intensive care units worldwidedand this trend is rising. 1e4 To provide an appropriate treatment for sepsis patients, the major challenge is not only the timely diagnosis of sepsis, but also the identification of the sepsis-causing agents. 4 A rapid detection of the causative pathogens enables early targeted antimicrobial therapy, which significantly increases survival rate, prevents subsequent complications, and reduces drugrelated side effects as well as medical expenses. 4e8 Despite extensive medical research in the field of sepsis, there are to date no fully satisfactory diagnostic approaches for a rapid, reliable, and sensitive identification of pathogens in the bloodstream. 9e12 Therefore, time-consuming, error-and contamination-prone blood cultures (BC) are still considered the standard of care for sepsis diagnostics, frequently leading to an inappropriate and delayed targeted therapy. 11,13

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confidence: 99%

Rapid Next-Generation Sequencing–Based Diagnostics of Bacteremia in Septic Patients

Grumaz

Hoffmann

Vainshtein

et al. 2020

The Journal of Molecular Diagnostics

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“…Although the third-generation sequencing such as the PacBio [7] and 10X [8,28] are now available, the majority of existing genome assemblies are currently derived from short-read based technologies. With the goal of evaluating genome assembly quality by assessing short read coverage distribution, we identi ed 1,156 HC and 15,034 LC regions in tomato genome assembly SL2.50 [25].…”

Section: Discussionmentioning

confidence: 99%

Impact of short-read sequencing on the misassembly of a plant genome

Wang

Meng

Moore

et al. 2020

Preprint

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Background Availability of plant genome sequences has led to significant advances. However, with few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues that could significantly impact any downstream analysis of plant genomes. In tomato for example, 0.6% (5.1 Mb) and 9.7% (79.6 Mb) of short-read based assembly had significantly higher and lower coverage compared to background, respectively. Results To understand what the causes may be for such uneven coverage, we first established machine learning models capable of predicting genomic regions with variable coverages and found that high coverage regions tend to have higher simple sequence repeat and tandem gene densities compared to background regions. To determine if the high coverage regions were misassembled, we examined a recently available tomato long-read based assembly and found that 27.8% (1.41 Mb) of high coverage regions were potentially misassembled of duplicate sequences, compared to 1.4% in background regions. In addition, using a predictive model that can distinguish correctly and incorrectly assembled high coverage regions, we found that misassembled, high coverage regions tend to be flanked by simple sequence repeats, pseudogenes, and transposon elements. Conclusions Our study provides insights on the causes of variable coverage regions and a quantitative assessment of factors contributing to plant genome misassembly when using short reads.

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“…Second, the fragmentationfree procedure reduces the bias introduced during library preparation, 16 resulting in more uniform coverage (Supplemental Figure S2). Third, the capacity of nanoporebased HLA typing is scalable and may be readily scaled up to much larger numbers of pores, 29 and it does not require a significant capital investment for equipment.…”

Section: Discussionmentioning

confidence: 99%

Accurate Typing of Human Leukocyte Antigen Class I Genes by Oxford Nanopore Sequencing

Liu

Xiao

Hoisington-Lopez³

et al. 2018

The Journal of Molecular Diagnostics

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Oxford Nanopore Technologies' MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunologic compatibility between organ donors and recipients, but the platform's high error rate makes it challenging to type alleles with accuracy. We developed and validated accurate typing of HLA by Oxford nanopore (Athlon), a bioinformatic pipeline that i) maps nanopore reads to a database of known HLA alleles, ii) identifies candidate alleles with the highest read coverage at different resolution levels that are represented as branching nodes and leaves of a tree structure, iii) generates consensus sequences by remapping the reads to the candidate alleles, and iv) calls the final diploid genotype by blasting consensus sequences against the reference database. Using two independent data sets generated on the R9.4 flow cell chemistry, Athlon achieved a 100% accuracy in class I HLA typing at the two-field resolution.

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A world of opportunities with nanopore sequencing

Cited by 27 publications

References 29 publications

Rapid Next-Generation Sequencing–Based Diagnostics of Bacteremia in Septic Patients

Rapid Next-Generation Sequencing–Based Diagnostics of Bacteremia in Septic Patients

Impact of short-read sequencing on the misassembly of a plant genome

Accurate Typing of Human Leukocyte Antigen Class I Genes by Oxford Nanopore Sequencing

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