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The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Capillary sequencing was the first approach to successfully elucidating a nearly complete human genome. However, it became apparent that this methodology would be too complex, lengthy and expensive for commercial purposes. Therefore, since 2005, capillary sequencing has been gradually replaced by high-throughput (formerly “next generation”) sequencing technologies such as Illumina’s dye sequencing, pyrosequencing, and SMRT sequencing. All of these technologies continue to employ the basic shotgun strategy (i.e., parallelization and template generation via genome fragmentation). Other technologies are still emerging such as nanopore-based methodologies. Although nanopore sequencing technology is still being refined, its portability and potential capability of reading long DNA sequences are likely to be important in the future for whole-genome sequencing applications.
Personalized Medicine for Cancer: Introduction and Overview of the Book
Published in II-Jin Kim, Cancer Genetics and Genomics for Personalized Medicine, 2017
(4) Less expensive and complicated technologies Single-cell analysis is now available [30] and third-generation nanopore sequencing [17] may become a standard in PM. However, such technological advances tend to be too complicated and expensive to handle in a regular clinic. Currently, NGS is a useful and powerful technology for identifying (biomarker discovery and mechanism identification) and screening (finding known key mutations) genetic alterations, but most small hospitals and clinics are not equipped to use NGS. It requires an expensive machine and bioinformatics analysis skills. Although a Clinical Laboratory Improvement Amendments (CLIA)-certified lab can be used, cost and turnaround time are still big issues, as are accuracy and interpretation of the data. Therefore, a simple, fast, and cost-effective genetic analysis system for PM will need to be developed so that PM would be available not only in resource-rich countries.
DNA Methods in Veterinary Medicine
Published in Rebecca A. Krimins, Learning from Disease in Pets, 2020
Traditional testing for pathogens can be time-consuming and require specialized laboratories. Human medicine is beginning to move towards DNA-based testing that can be completed more quickly and with higher precision. The current limitations of DNA sequencing are related to speed, cost and sensitivity. Nanopore sequencing may partially solve these issues by providing a relatively low cost and rapid technology, that despite having a much higher error rate than traditional sequencing can overcome that limitation by creating consensus sequences from many individually erroneous reads. An interesting application of nanopore sequencing is that it can be used in the field to monitor infectious disease (e.g., cholera; Acharya et al., 2019) and Ebola (Quick et al., 2016), where sequences from the pathogen of interest are amplified by PCR and samples loaded and run on a nanopore flow cell. For cholera and other prokaryotes the regions applied are usually the 16s rRNA genes whose sequences vary between species and can serve as a reliable taxonomic biomarker (Cheng et al., 2018). The relative speed (as little as a few hours), cost and portability of nanopore sequencing is likely to see it become used in ERs and clinics in the near term. In veterinary medicine nanopore sequencing has been demonstrated as a tool to diagnose distemper virus (Peserico et al., 2019) and to monitor antibiotic resistance in a veterinary hospital (Kamathewatta, et al., 2019). As the technology continues to improve in both read accuracy and ease of use it is likely that nanopore sequencing will find many applications in veterinary medicine, environmental monitoring and biohazard identification, among others.
NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells
Published in mAbs, 2022
Hema Preethi Subas Satish, Kathleen Zeglinski, Rachel T. Uren, Stephen L. Nutt, Matthew E. Ritchie, Quentin Gouil, Ruth M. Kluck
We conclude that NAb-seq is ideally suited for quickly and cheaply generating high-accuracy antibody sequences from between 1 and 24 hybridoma cell lines or single B cells. The low up-front cost of the minION nanopore sequencing instrument (US$1000), low cost per run (~US$30 per sample), universal protocol, flexible throughput and quick turnaround enable NAb-seq to be performed in-house and easily integrated into existing workflows. For much larger sample numbers (e.g., hundreds or thousands of hybridomas or single B cells using 10X single-cell approaches), targeted methods like RAGE-Seq which sequence only heavy/light chain amplicons become more efficient.29 Still, the computational error correction (consensus) approach implemented in NAb-seq could also be applied to targeted methods to generate high accuracy consensus sequences.
New Insights of Corynebacterium kroppenstedtii in Granulomatous Lobular Mastitis based on Nanopore Sequencing
Published in Journal of Investigative Surgery, 2022
Xin-Qian Li, Jing-Ping Yuan, Ai-Si Fu, Hong-Li Wu, Ran Liu, Tian-gang Liu, Sheng-Rong Sun, Chuang Chen
Of note, the positive rate of C. kroppenstedtii vary considerably in different reports, mainly due to detection techniques and samples. It is difficult to culture C. kroppenstedtii in conventional medium [11]. Although Gram staining can recognize Gram-positive Corynebacterium within cystic vacuoles, typical vacuoles are poorly studied [12,13]. Moreover, these aspects need to be investigated in conjunction with other morphological and genetic approaches [14]. Over the past decade, gene sequencing has tremendously improved microbial identification in GLM. The sequencing technologies for bacterial detection in GLM include qPCR, Sanger sequencing and next-generation sequencing [5,15,16]. However, most of the previous studies used formalin-fixed and paraffin-embedded (FFPE) tissues for sequencing. These samples showed a low detection rate of C. kroppenstedtii due to DNA degradation caused by paraffin [5]. While in fresh samples, the detection rate of C. kroppenstedtii in the early stage of GLM were rarely reported [16]. Nanopore sequencing is a third-generation sequencing technology. This method has greatly improved the detection speed and read length. Indeed, it allows for sequencing of complete DNA/RNA sequences directly without amplification [17,18], and this technology has been widely applied in the area of microbiology [19,20].
Pharmacogenomics in the era of next generation sequencing – from byte to bedside
Published in Drug Metabolism Reviews, 2021
Laura E. Russell, Yitian Zhou, Ahmed A. Almousa, Jasleen K. Sodhi, Chukwunonso K. Nwabufo, Volker M. Lauschke
These drawbacks of SRS were tackled by the introduction of long-read sequencing (LRS), which is capable of covering up to 100 Kb (van Dijk et al. 2018). Two major platforms are available for LRS application: PacBio single-molecule real-time sequencing (SMRT Seq) and nanopore sequencing. SMRT Seq is an optical method in which the integration of fluorescently labeled nucleotides into a nascent nucleic acid chain by an immobilized DNA polymerase is monitored in real time (Eid et al. 2009). In contrast, nanopore sequencing quantifies the fluctuations in ionic currents that differ between nucleotides upon translocation of a nucleic acid chain through a nanopore (Feng et al. 2015). While both methods can overcome the technical limitations of SRS, the substantially higher cost and longer turnaround times still limit their routine use for clinical applications.