Laboratory techniques to study the cellular and molecular processes of disorders
Louis-Philippe Boulet in Applied Respiratory Pathophysiology, 2017
Gene sequencing, the task of determining the nucleotide order of a gene, is analogous to the spelling of a word and is made possible with PCR. Briefly, gene sequencing begins with the hybridization of a primer complementary to the fragment to be sequenced. The addition of deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP) terminates the elongation process and reveals the last incorporated nucleotide. By comparing the sequence of a gene of interest between the affected and nonaffected subjects, differences can be subsequently investigated for causality. If the mutation occurs in exons, it could potentially modify the structure and function of the translated protein. Alternatively, a mutation in the regulatory regions of a gene (e.g., 3′untranslated region [UTR], 5′UTR or promoter) could affect protein expression. High throughput sequencing technologies have also been developed to reduce sequencing effort by targeting specific genetic elements in the genome for sequencing. While exome sequencing focuses on sequencing the coding portions, next-generation sequencing (NGS) targets exomes and regulatory regions. For example, by using whole-exome sequencing in one pedigree with asthma, researchers have identified 10 novel nonsynonymous variants found only in affected individuals in the pedigree [33]. These technologies and available platforms have been described schematically and comprehensively elsewhere [34].
RNA-seq Analysis
Altuna Akalin in Computational Genomics with R, 2020
With the advent of the second-generation (a.k.a next-generation or high-throughput) sequencing technologies, the number of genes that can be profiled for expression levels with a single experiment has increased to the order of tens of thousands of genes. Therefore, the bottleneck in this process has become the data analysis rather than the data generation. Many statistical methods and computational tools are required for getting meaningful results from the data, which comes with a lot of valuable information along with a lot of sources of noise. Fortunately, most of the steps of RNA-seq analysis have become quite mature over the years. Below we will first describe how to reach a read count table from raw fastq reads obtained from an Illumina sequencing run. We will then demonstrate in R how to process the count table, make a case-control differential expression analysis, and do some downstream functional enrichment analysis.
Molecular Biology in Colorectal Adenoma and Adenocarcinoma
Peter Sagar, Andrew G. Hill, Charles H. Knowles, Stefan Post, Willem A. Bemelman, Patricia L. Roberts, Susan Galandiuk, John R.T. Monson, Michael R.B. Keighley, Norman S. Williams in Keighley & Williams’ Surgery of the Anus, Rectum and Colon, 2019
Advances in DNA sequencing technology have resulted in massive increases in the speed of sequencing and volume of throughput, as well as dramatic decreases in cost. This has allowed the analysis of large numbers of adenomas and cancers, and led to the identification of many genes potentially implicated in colorectal carcinogenesis. One example of this is a study in which somatic mutations were identified in over 800 genes, of which 140 were thought to contribute to the cancer phenotype.2 It is currently thought that, on average, an advanced cancer contains about 20 mutated genes that probably contribute to the malignant phenotype, and over ten times as many other genes that are mutated as a result of the derangements in the tumour, but do not play a part in carcinogenesis.
Gastric protective effect of Alpinia officinarum flavonoids: mediating TLR4/NF-κB and TRPV1 signalling pathways and gastric mucosal healing
Published in Pharmaceutical Biology, 2023
Kaiwen Lin, Tang Deng, Huijuan Qu, Hongya Ou, Qifeng Huang, Bingmiao Gao, Xiaoliang Li, Na Wei
High-throughput sequencing is a transcriptome sequencing technique. Through sample preparation, library establishment, mRNA sequencing operation and data screening and analysis, the mechanism of candidate genes or potential molecular signal pathways are identified (Hrdlickova et al. 2017; Yang et al. 2020). According to GO enrichment analysis (Figure 5(B)), we speculated that F.AOH might improve or inhibit GU occurrence by enriching and regulating immune response, participating in the oxidative stress process, protecting the integrity of gastric mucosa, and regulating cell adhesion. Furthermore, based on KEGG pathway enrichment analysis results (Figure 5(D)), we speculated that F.AOH could improve the immune function of rats, control metabolic disorders, enhance the information transmission of biological cascade reactions, regulate cell proliferation, migration, apoptosis, protect the integrity of gastric mucosa, and prevent GU from developing to severe or even canceration. Then, Toll-like receptors signalling pathways, regulation of inflammatory mediators TRP channels, and PI3K-Akt signalling pathway in several key genes and proteins for validation were chosen. Finally, we found FAOH treatment can promote GU anti-inflammatory analgesic action, promote healing of ulcers of tissue repair, and epithelial cell proliferation and migration to improve the cure rate of GU. The pathological mechanism of these imbalances is closely related to GU occurrence and development (Tarnawski and Ahluwalia 2012).
Understanding the extent of the diagnostic potential of coagulation factors
Published in Expert Review of Molecular Diagnostics, 2020
Emmanuel J. Favaloro, Giuseppe Lippi
Until recently, Sanger sequencing of specific candidate genes has been the only method available for molecular diagnosis of coagulation factors [7]. However, this approach is time-consuming, costly and requires some pre-identification of the proposed candidate gene(s). Thus, factor assays or family studies would have already needed to be performed, in order to identify the deficient/defective coagulation protein and therefore to identify which gene (and what primers) would be needed for specific molecular interrogation. Increasingly, high-throughput sequencing (HTS) is being performed to allow simultaneous and rapid investigation of multiple genes at a manageable cost. This newer technology includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, and is revolutionizing the genetic diagnosis of human diseases, including those associated with bleeding disorders [7]. Some relevant examples are provided in the references and Table 1. Nevertheless, its implementation remains limited in diagnostics, and while it is increasingly progressed into clinical practice, its main utility remains in the research field.
A Review of Next-Generation Sequencing (NGS): Applications to the Diagnosis of Ocular Infectious Diseases
Published in Seminars in Ophthalmology, 2019
Lina Ma, Frederick A. Jakobiec, Thaddeus P. Dryja
Next-generation sequencing (NGS) is a newer method for evaluating infections. It is a high throughput sequencing method using a massively parallel sequencing platform.16–18 NGS sequences millions of small DNA fragments simultaneously over the course of a few hours. Each fragment is 150 to 500 bases length depending on the type of NGS platform used (Table 2). Most of the sequenced fragments will be human DNA fragments from the patient’s cells; only a small minority of the fragments (0.1–8%) will be derived from the pathogenic organism.19 With this technology, one can sequence DNA fragments from a biopsy of infected ocular tissue or from intraocular fluid. Laser-microdissected formalin-fixed and paraffin-embedded (FFPE) tissue specimens can also be used for NGS.16–19Table 3 provides a list of reference laboratories to which specimens can be sent for NGS analysis at a reasonable cost, which is often covered by standard health insurances.
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