The Third Revolution in Sequencing Technology

Dijk, Erwin L. van; Jaszczyszyn, Yan; Naquin, Delphine; Thermes, Claude

doi:10.1016/j.tig.2018.05.008

Cited by 843 publications

(587 citation statements)

References 73 publications

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“…PRRSV ORF5 shows extensive genetic diversity and has been used for providing insight into PRRSV epidemiology, however it is only 5% of the whole genome, thus 95% of the genomic information remains for prediction of genetic variation. Whole genome sequencing is greatly needed to provide a more complete picture of the virus [67,68], which is now gradually becoming more feasible with the rapid development and innovation of new sequencing technologies [69,70]. Oxford Nanopore direct RNA sequencing (DRS) is revolutionary for sequencing RNA viral genomes, since it can sequence the RNA directly, allowing for detection of methylation sites and decreasing bias inherent in reverse transcription and PCR amplification of samples prior to sequencing, and it can generate long reads, allowing for the elucidation of recombination events [71].…”

Section: Discussionmentioning

confidence: 99%

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

Tan

Dvorak

Murtaugh

2019

Viruses

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Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 10 4 to 10 6 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks.2 of 18 against a new introduction from outside the farm indicates a need for enhanced biosecurity, while a re-break of a resident strain suggests better strategies for an elimination or vaccination program are needed. Another big challenge for PRRS control is that PRRSV vaccine is not completely effective at preventing and controlling infection due to the high genetic diversity of the virus, thus outbreaks still occur in vaccinated herds [13][14][15][16]. A diagnostic method which can provide genetic information about the strain causing infection would allow for identification of potential reasons for vaccination failure, such as limited cross-protection due to high genetic divergence from vaccine [17], or in the case of genetic similarity to vaccine, perhaps a reversion to virulence (escape mutation) of the vaccine itself [18]. In addition to the clinical challenges mentioned above, PRRSV has widely divergent genetic lineages and is a rapidly evolving pathogen with novel variants which seem to be more divergent and virulent than those in the past [10,[19][20][21]. The continuous emergence of new virulent strains causes unexpected devastating outbreaks, such as the severe outbreaks of HP-PRRSV in China and MN184 and NADC30 outbreaks in the United States [22][23][24][25]. The increasing incidence of co-infections of multiple strains further complicates PRRS diagnosis and control [26]. Hence, diagnostic tools that can provide more genetic information are extremely important for investigation, prevention, and control strategies for PRRSV outbreaks.Prompt detection of pathogens during an outbreak is essential for efficient disease control. Real-time quantitative...

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Section: Discussionmentioning

confidence: 99%

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

Tan

Dvorak

Murtaugh

2019

Viruses

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“…Recently, thirdgeneration of sequencing (TGS) technologies have been developed and widely used. [35] By applying this long read-length technology, the sequence and integrity of these dicistron mRNAs can be detected. The cryptic promoters and splicing sites can also be identified, because both of them generate abnormally truncated transcripts (see Box Figure 2).…”

Section: The Specificity Guarantees the Quality Of Ires Identificationmentioning

confidence: 99%

A New Way to Discover IRESs in Pathology or Stress Conditions? Harnessing Latest High‐Throughput Technologies

et al. 2020

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The cellular internal ribosomal entry site (IRES) is one of the most important elements to mediate cap‐independent translational initiation, especially under conditions of stress and pathology. However, a high‐throughput method to discover IRESs in these conditions is still lacking. Here, a possible way IRES long‐read sequencing based on the latest high‐throughput technologies is proposed to solve this problem. Based on this design, diversity and integrity of the transcriptome from original samples can be kept. The micro‐environment that stimulates or inhibits IRES activity can also be mimicked. By using long read‐length sequencing technology, additional experiments that are essential for ruling out the cryptic promoters or splicing events in routine IRES identification processes can be circumvented. It is hoped that this proposed methodology may be adopted for IRES element discovery, hence uncovering the full extent of the role of IRESs in disease, development, and stress. Also see the video abstract here https://youtu.be/JuWBbMzWXS8

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“…Nanopore sequencing technology was made public by Oxford Nanopore Technologies (ONT) in 2014. When DNA or RNA module translocates through a nanopore, an ionic current will be produced from a constant voltage bias, and then a change in the ionic current can be observed (van Dijk et al, 2018). Nucleic acid bases can then be basecalled by built-in or the third-party basecallers.…”

Section: Introductionmentioning

confidence: 99%

“…Nucleic acid bases can then be basecalled by built-in or the third-party basecallers. When compared to the next-generation sequencing (NGS) methods, nanopore sequencing technology has advantages such as real time, long reads, short turnaround time, and simple sample preparation procedures (van Dijk et al, 2018;Ameur et al, 2019). In particular, the ONT platform MinION, also characterized by portability and low cost in addition to the above advantages, can well handle rapid pathogen detection in the field (McIntyre et al, 2016;Castro-Wallace et al, 2017;Johnson et al, 2017;Edwards et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

First Identification of Human Adenovirus Subtype 21a in China With MinION and Illumina Sequencers

Han

Zhu

et al. 2020

Front. Genet.

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The Third Revolution in Sequencing Technology

Cited by 843 publications

References 73 publications

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

A New Way to Discover IRESs in Pathology or Stress Conditions? Harnessing Latest High‐Throughput Technologies

First Identification of Human Adenovirus Subtype 21a in China With MinION and Illumina Sequencers

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