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The Brave New World of Genomics
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Sandra García Herrero, Blanca Simon Frances, Cristian Perez-Garcia, Javier Garcia-Planells
The impressive and continual development of genomic technologies in recent years has been leading this genomic revolution. Since 2005, the year in which Roche began to commercialize the first NGS sequencer, an important race started to reach the goal of clinical diagnosis. Two years later, a new technology, Illumina, and thereafter other competitors such as SOLID and Ion Torrent, currently marketed by Thermo Fisher, joined the bandwagon [5]. Third-generation sequencing technologies are based on single-molecule sequencing, a new approach that avoids the bias of PCR amplification and produces longer sequences. Although a clear advantage is associated with this new technology, the caveat is the loss of sensitivity, a key aspect in diagnosis [6–8]. The recently reported and promising technology, fourth-generation sequencing, centered around the in situ reading of nucleic acid sequences within tissues and cells, exploits previous NGS chemistries [9]. This technology pledges to become an essential tool for the implementation of personalized medicine [10,11].
Next-Generation Sequencing (NGS) for Companion Diagnostics (CDx) and Precision Medicine
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Il-Jin Kim, Mendez Pedro, David Jablons
All the sequencing systems mentioned above produce short sequence reads ranging from 25–1000 bp.18 Compared to the first version of an NGS system about 10 years ago, the read length has been improved in general. However, it is still not enough to detect complex structural variants with such short reads. If multiple nucleotide variants at consecutive sequences (i.e., CT variants in one allele vs. C variant in one allele and T variant in another allele) are shown in one sample, it is hard to call correct genotypes or variants in regular short-read NGS without phasing.35 A long-read sequencing can provide correct genotypes because it facilitates de novo assembly with phasing information.35,36 Thus, it is a major goal to make long reads when developing new NGS or third-generation sequencing systems. Moreover, long-read or third-generation sequencing removes clonal amplification and can thereby avoid the PCR-related bias.24 It can also detect native DNA directly rather than secondary signals from DNA incorporation.24,37 The following two systems are leading sequencing technologies for long-read or third-generation sequencing.
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].
Cytogenetic and molecular genetic methods for chromosomal translocations detection with reference to the KMT2A/MLL gene
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Nikolai Lomov, Elena Zerkalenkova, Svetlana Lebedeva, Vladimir Viushkov, Mikhail A. Rubtsov
Long-read sequencing is a more convenient method for detecting structural variants, which refer to genomic changes that are conventionally larger than 50 nt in size, including deletions, duplications, insertions, inversions, and translocations [99,102,103]. Besides, due to substantial length of reads, third-generation sequencing was successfully used to close the gaps in the primary assembly of the human genome indeterminable by short-read methodology [104] and was proven to be effective in chromosomal translocations identification [105]. However, both third-generation sequencing technologies still have limitations that prevent their widespread use in clinical practice. The PacBio method is very expensive and has a lower throughput than other methods, making it relatively time-consuming, especially when deep sequencing is necessary. The Oxford Nanopore method returns a very high error rate (up to 15%), and the expensive nanopore membranes necessary for this technique have a limited shelf life before they must be replaced [106]. Therefore, we will not consider third-generation sequencing any further in this review.
Proteomics and the microbiome: pitfalls and potential
Published in Expert Review of Proteomics, 2019
Huafeng Lin, Qing-Yu He, Lei Shi, Mark Sleeman, Mark S. Baker, Edouard C. Nice
The most important benefit of using sequencing approaches to study the microbiome is that they give a more comprehensive profiling of the species present in a sample compared with traditional culture methods [10, 47]. Sequencing technology has continuously advanced over the past 30 years since the First-generation DNA sequencing was reported in 1977 by Sanger [48]. Next-generation sequencing technologies have revolutionized high throughput sequencing in biological sciences. These supplanted the Sanger method by providing dramatic reduction in cost, albeit at the expense of read lengths [49]. Third-generation sequencing technology, in which nucleotide sequences are read at the single molecule level, in contrast to previous methods in which long strands of DNA were broken into small segments and the sequences determined by amplification and synthesis, has many merits, including high throughput, high speed, long read lengths, and drastically reduced cost [50]. DNA sequencing has now entered the era of single-molecule nanopore technology since Nanopore-based sequencing, a fourth-generation DNA sequencing technology, was reported at the end of 2012 [51, 52] and has suggested to enable rapid and reliable sequencing of a whole human genome at a cost of below $1000, ultimately possibly even less than $100 (Table 1).