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Genetic and genomic investigations
Published in Angus Clarke, Alex Murray, Julian Sampson, Harper's Practical Genetic Counselling, 2019
The Sanger method of DNA sequencing was originally performed manually. Automated methods were developed, which still depended upon a prior amplification step of the sample DNA. Newer methods, often known as ‘next-generation’ or ‘high-throughput’ sequencing, have been developed that determine the sequence of very large numbers of single DNA molecules in parallel, without any amplification. These methods differ in the length of sequence that they can determine and the chance of introducing error. Some methods detect charge differences as large molecules move across a boundary; others depend upon changes associated with DNA synthesis. Methods include pyrosequencing, nanopore sequencing and ion semiconductor sequencing. The cost per base of sequencing has fallen dramatically, so that applications of sequencing that would have been inconceivable 20 years ago are now commonplace both in research and, increasingly, in diagnostics.
Understanding the extent of the diagnostic potential of coagulation factors
Published in Expert Review of Molecular Diagnostics, 2020
Emmanuel J. Favaloro, Giuseppe Lippi
Additional emerging technologies, set to include the field of hemostasis, include short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing [10]. The main current applications for such methods include targeted resequencing, exome sequencing, transcriptome sequencing, small genome sequencing, along with liquid biopsy. The main advantage of the system is the low relative cost per base [10]. Drawbacks include relatively low throughput and challenges to wider implementation. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of hematological disease mechanisms is becoming possible [10]. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will also expectedly continue the rapid evolution of this area [10]. As an example, current nanopore technology uses protein-based nanopores that are structurally precise, easily modified and able to be engineered and produced on a large scale, thus enabling accurate analysis of proteins at the single-molecule level. However, a large degree of development is required before solid-state nanodevice DNA sequencing becomes mainstream.
Use of whole genome sequencing in surveillance of drug resistant tuberculosis
Published in Expert Review of Anti-infective Therapy, 2018
Ruth McNerney, Matteo Zignol, Taane G Clark
NGS has been used in numerous TB studies to investigate transmission dynamics, outbreaks, and explore patterns of resistance with over 10,000 MTB genomes sequenced [15,17,18,43–46]. Two platforms, Illumina sequencing (Illumina, San Diego, USA) [47] and Ion Torrent or Ion semiconductor sequencing (Ion Torrent Inc., USA, marketed by Thermo Fisher Scientific) [48], have been successfully used to analyze drug-resistant MTB [49]. Studies in the United Kingdom, the United States, and Italy suggest that NGS may be faster and more cost effective for mycobacterial identification and drug resistance testing than traditional methods [50–54]. However, sequencing requires amounts of MTB DNA that are rarely found in clinical specimens and isolation and culture is needed to produce sufficient template for reliable WGS, a process that can take weeks [55]. Efforts to sequence directly from sputum samples are ongoing and proof of principle has been achieved [53,56–58]. The use of a sample enrichment method where the MTB DNA was selectively captured utilizing biotinylated RNA baits enabled WGS to be performed on all 20 smear-positive sputum samples tested and one sample that was smear negative [57]. The methodology has already been used to guide treatment for drug-resistant TB in the United Kingdom [58]; however, the high cost of the reagents needed may constrain its application in resource-limited settings where TB is endemic [57]. In a separate study, also conducted in the UK, a more rapid and less expensive extraction procedure was found to yield genome sequence data sufficient to predict drug resistance for 24 of 40 sputum samples tested within 48 h [53]. Efforts are continuing to improve and develop methodologies that are both affordable and sufficiently robust for use in surveillance studies. New sequencing technologies are also being developed that promise faster results and lower running costs and some have the potential to sequence from reduced amount of DNA. A portable sequencing device, the MinION sequencer (Oxford Nanopore Technologies, Oxford, UK), has been shown in a feasibility study to be capable of sequencing MTB to provide information on drug resistance [59].