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Next-generation sequencing applications. Schematogram depicting the different methods for transcriptomic, miRNomic, epigenomic and genomic studies. 

Next-generation sequencing applications. Schematogram depicting the different methods for transcriptomic, miRNomic, epigenomic and genomic studies. 

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Article
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Within just a few years, the new methods for high-throughput next-generation sequencing have generated completely novel insights into the heritability and pathophysiology of human disease. In this review, we wish to highlight the benefits of the current state-of-the-art sequencing technologies for genetic and epigenetic research. We illustrate how...

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... the following paragraphs, we want to provide an overview on different NGS applications, starting with genomic sequencing (Figure 1). DNA sequencing by NGS can be applied within different applications, such as partial-exome (PES), whole-exome (WES) or whole-genome sequencing (WGS). ...

Citations

... The development of Next Generation Sequencing (NGS), which is also referred to as deep-sequencing, or high-throughput sequencing, has provided a set of diverse modern technologies with applicability to the study of DNA, RNA and proteins [197]. In particular, RNA-seq methodologies allow for the determination of the sequence of an overwhelming amount of different RNA molecules in a massively parallel way [197,198]. ...
... The development of Next Generation Sequencing (NGS), which is also referred to as deep-sequencing, or high-throughput sequencing, has provided a set of diverse modern technologies with applicability to the study of DNA, RNA and proteins [197]. In particular, RNA-seq methodologies allow for the determination of the sequence of an overwhelming amount of different RNA molecules in a massively parallel way [197,198]. ...
Article
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A long scientific journey has led to prominent technological advances in the RNA field, and several new types of molecules have been discovered, from non-coding RNAs (ncRNAs) to riboswitches, small interfering RNAs (siRNAs) and CRISPR systems. Such findings, together with the recognition of the advantages of RNA in terms of its functional performance, have attracted the attention of synthetic biologists to create potent RNA-based tools for biotechnological and medical applications. In this review, we have gathered the knowledge on the connection between RNA metabolism and pathogenesis in Gram-positive and Gram-negative bacteria. We further discuss how RNA techniques have contributed to the building of this knowledge and the development of new tools in synthetic biology for the diagnosis and treatment of diseases caused by pathogenic microorganisms. Infectious diseases are still a world-leading cause of death and morbidity, and RNA-based therapeutics have arisen as an alternative way to achieve success. There are still obstacles to overcome in its application, but much progress has been made in a fast and effective manner, paving the way for the solid establishment of RNA-based therapies in the future.
... Platforms designed over the years are 454 pyrosequencing, Illumina, SOLiD, the Polonator, HeliScope Single Molecule Sequencer, Ion Torrent, and PacBio RS. However, NGS involves the fulfillment of five distinct steps; (1) fragmentation, (2) library preparation, (3) massive parallel sequencing, (4) bioinformatic analysis, and (5) interpretation and variant/mutation annotation [72][73][74][75]. ...
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Background: PGT-based NGS revolutionized the field of reproductive medicine, becoming an integrated component within current assisted reproductive technology (ART) protocols. Methods: We searched the literature published in the last half a decade in four databases (PubMed/Medline, ISI Web of Knowledge, ScienceDirect, and Scopus) between 2018 and 2022. Results: A total of 1388 articles were filtered, from which 60 met, initially, the eligibility criteria, but only 42 were included (≥100 patients/couples-62,465 patients and 6628 couples in total) in the present mini-review. In total, forty-two (70.0%) reported reproductive outcomes, while eighteen (30.0%) had distinct objectives. Furthermore, n = 1, 1.66% of the studies focused on PGT, n = 1, 1.66% on pre-implantation genetic testing for monogenic disorders (PGT-M), n = 3, 5.0% on pre-implantation genetic testing for structural rearrangements (PGT-SR) and n = 55, 91.66% on pre-implantation genetic testing for aneuploidies (PGT-A). Conclusions: PGT using NGS proved to be an excellent companion that folds within the current ascending tendency among couples that require specialty care. We strongly encourage future studies to provide a systematic overview expanded at a larger scale on the role of the PGT-NGS.
... The term NGS includes a group of technologies, also called massively parallel sequencing, that share the ability to simultaneously analyze multiple genomic regions through data capture from millions of sequencing reactions. [4][5][6][7] This technique is linked with bioinformatic tools which are essential for analyzing the vast amount of generated data. 8 These data can be used to support patient management. ...
Article
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Next-generation sequencing (NGS) has been implemented in clinical oncology for diagnosis, prognosis, and therapeutic guidance. Among the various NGS applications in molecular oncology, we focused on the following topics: laboratory standards for targeted gene panels (somatic mutations) and therapeutic guidance based on NGS of lung cancer and rare cancers, namely sarcomas and cancers of unknown primary. Multiple quality control checkpoints should be addressed in the pre-analytical phase for good quality and interpretation of the NGS results. It includes tumor size and cellularity, tissue processing and decalcification, tumor fraction, tumor viability, fixatives, and staining. Communication between clinicians and laboratory support is also essential. In lung cancer, all patients with non-squamous non-small cell lung cancer should be tested with a NGS panel, and it should include not only genes with approved targeted therapies (ALK, BRAF, EGFR, MET, NTRK, RET, and ROS1) but also genes with potentially actionable genomic alterations (HER2 and KRAS). Since there is a lack of extensive knowledge regarding the use of NGS in rare tumors performing comprehensive genomic profiling, NGS panels to better manage the disease are recommended. Moreover, other patients with other incurable solid tumors may benefit from being included in biomarker-driven clinical trials. Multidisciplinary tumor boards with the participation of experts with the ability to integrate genomic profiling data are essential to tailor the best strategy for each patient. Considering that there are no national guidelines, this article aims to guide laboratory and clinical practice for the use of NGS in the context of lung cancer, rare tumors, and cancer of unknown primary in Portugal.
... Reported to be expressed from unicellular eukaryotic organism to humans, lncRNAs show tissue/cell/stage specific differential expression, are not well conserved, exhibit different subcellular localization and shows enormous diversity in its role and mechanism to regulate expression of protein coding genes (3)(4)(5)(6)(7). With the recent developments in deep sequencing technologies, their repertoire is exponentially increasing however, biological significance and characterization of most of lncRNA remains elusive (8,9). Some wellstudied lncRNAs are reported to be the key players in distinct processes like dosage compensation, genomic imprinting, epigenetic regulator, pluripotency, post transcription regulator of mRNA, modulator of stability/translation of mRNA (10)(11)(12)(13)(14)(15)(16). ...
Article
Full-text available
Long non-coding RNA has emerged as a key regulator of myriad gene functions. One such lncRNA mrhl, reported by our group, was found to have important role in spermatogenesis and embryonic development in mouse. Recently, its human homolog, Hmrhl was shown to have differential expression in several type of cancers. In the present study, we further characterize molecular features of Hmrhl and gain insight into its functional role in leukemia by gene silencing and transcriptome-based studies. Results indicate its high expression in CML patient samples as well as in K562 cell line. Silencing experiments suggest role of Hmrhl in cell proliferation, migration & invasion. RNA-seq and ChiRP-seq data analysis further revealed its association with important biological processes, including perturbed expression of crucial TFs and cancer-related genes. Among them ZIC1, PDGRFβ and TP53 were identified as regulatory targets, with high possibility of triplex formation by Hmrhl at their promoter site. Further, overexpression of PDGRFβ in Hmrhl silenced cells resulted in rescue effect of cancer associated cellular phenotypes. In addition, we also found TAL-1 to be a potential regulator of Hmrhl expression in K562 cells. Thus, we hypothesize that Hmrhl lncRNA may play a significant role in the pathobiology of CML.
... FACS, which enables the sorting of single cells, allows the enrichment of specific cells to generate highresolution gene expression and transcriptional maps (Kambale et al., 2019). NGS and RNA sequencing (RNA-seq) technologies enable the large-scale DNA and RNA sequencing of the entire genome and transcriptome, respectively, providing an unbiased and comprehensive view of biological systems towards understanding genomic function (Frese et al., 2013;Alfaro et al., 2019;Stark et al., 2019). Examples of NGS platforms include the Illumina HiSeq, Genome Analyzer Systems, 454 Genome Sequencer FLX Titanium System, the Helicos HeliScope, the SOLiD sequencing platform and the Ion Torrent Sequencing platform. ...
Article
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Biomanufacturing is a key component of biotechnology that uses biological systems to produce bioproducts of commercial relevance, which are of great interest to the energy, material, pharmaceutical, food, and agriculture industries. Biotechnology-based approaches, such as synthetic biology and metabolic engineering are heavily reliant on “omics” driven systems biology to characterize and understand metabolic networks. Knowledge gained from systems biology experiments aid the development of synthetic biology tools and the advancement of metabolic engineering studies toward establishing robust industrial biomanufacturing platforms. In this review, we discuss recent advances in “omics” technologies, compare the pros and cons of the different “omics” technologies, and discuss the necessary requirements for carrying out multi-omics experiments. We highlight the influence of “omics” technologies on the production of biofuels and bioproducts by metabolic engineering. Finally, we discuss the application of “omics” technologies to agricultural and food biotechnology, and review the impact of “omics” on current COVID-19 research.
... Valuable new pieces of information about genomic drivers of cancer onset and progression across several anatomical locations have been highlighted thanks to the application of nextgeneration sequencing (NGS) techniques to discovery projects on large-scale cancer genomics (12). Unlike traditional Sanger sequencing, the NGS has the ability to sequence, very efficiently and at high throughput, gigabases of DNA (13). The majority of NGS approaches rely on DNA template preparation, sequencing and imaging, and data analysis. ...
Article
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During the last century, cancer biology has been arguably one of the most investigated research fields. To gain deeper insight into cancer mechanisms, scientists have been attempting to integrate multi omics data in cancer research. Cancer genomics, transcriptomics, metabolomics, proteomics, and metagenomics are the main multi omics strategies used currently in the diagnosis, prognosis, treatment, and biomarker discovery in cancer. In this review, we describe the use of different multi omics strategies in cancer research in the African continent and discuss the main challenges facing the implementation of these approaches in African countries such as the lack of training programs in bioinformatics in general and omics strategies in particular and suggest paths to address deficiencies. As a way forward, we advocate for the establishment of an “African Cancer Genomics Consortium” to promote intracontinental collaborative projects and enhance engagement in research activities that address indigenous aspects for cancer precision medicine.
... Embedded in a long string spanning several billion characters, drawn from a set of genetic alphabets, the genomic (A), cytosine (C), guanine (G) and thymine (T), within the genomic sequence, and the process is widely known as sequencing. Next Generation Sequencing (NGS) involves massively parallel sequencing of genetic data with high throughput, while offering an unparalleled interrogation of the genome, throwing deeper insight into the functional and structural investigation of genetic data [1,2]. ...
Article
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Genome Informatics (GI) involves accurate computational investigations of strongly correlated subsystems that demands inter-disciplinary approaches for problem solving. With the growing volume of genomic sequencing data at an alarming rate, High Performance Computing (HPC) solutions offer the right platform to address the computational needs. GI requires algorithm-architecture co-design of parallel and accelerated biocomputing involving reconfigurable hardware like FPGAs and graphics accelerators or GPUs, to bridge the gap between growing data volumes and compute capabilities. Such platforms offer high degrees of parallelism and scalability, while accelerating the multi-stage GI computational pipeline. Amidst such high computing power, it is the choice of algorithms and implementations in the entirety of the GI pipeline that decides the precision of bio-computing in revealing biologically relevant information. Through this paper, we present ReneGENE-GI, an innovatively engineered GI pipeline. This paper details the performance analysis of ReneGENE-GI’s Comparative Genomics Module (CGM), the compute intensive stage of the pipeline. This module comes in two flavours, designed to run on GPUs and FPGAs respectively, hosted on HPC platforms. The pipeline uses a very efficient reference indexing algorithm based on the dynamic Monotonic Minimal Perfect Hashing Function (MMPH), allowing an absolute indexing for the reference genome, thus avoiding heuristics. Alignment time for our FPGA version is about one-tenth the time taken by our single GPU implementation, which itself is 2.62x faster than CUSHAW2-GPU (the GPU CUDA implementation of CUSHAW). With the single-GPU implementation demonstrating a speed up of 150+ x over standard heuristic aligners in the market like BFAST, the FPGA version of our CGM is several orders faster than the competitors, offering precision over heuristics.
... Reported to be expressed from unicellular eukaryotic organism to humans, lncRNAs show tissue/cell/stage specific differential expression, are not well conserved, exhibit different subcellular localization and shows enormous diversity in its role and mechanism to regulate expression of protein coding genes (3)(4)(5)(6)(7). With the recent developments in deep sequencing technologies, their repertoire is exponentially increasing, however biological significance and characterization of most of lncRNA remains elusive (8,9). Some lncRNAs which are well studied are reported to be the key players in distinct processes like dosage compensation, genomic imprinting, epigenetic regulator, pluripotency, post transcription regulator of mRNA, modulator of stability/translation of mRNA (10)(11)(12)(13)(14)(15)(16). ...
... Briefly, the matrigel membrane was first rehydrated by adding 500μl of warm (37˚C) media (RMPI-1640 with 10% FBS) in both the chambers of the insert and incubating it for 2 hrs at 37˚C. After rehydration 750 μL RPMI-1640 with 20% FBS, was added to the lower compartment of the invasion chamber while K562 cells (2.5x10 5 , with/without silenced Hmrhl) 8 were suspended in 500μL serum-free RPMI-1640 and were seeded in the upper compartment of the invasion chambers and incubated for 48 hrs. After 48 hrs, cells that invaded to the bottom chamber were analyzed by manual cell counting using 0.4% trypan blue as well as by CCK8 assay. ...
Preprint
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Long non-coding RNA has emerged as a key regulator of myriad gene functions. One such lncRNA mrhl, reported by our group, was found to be a regulator of SOX8 , Wnt-signalling along with an important role in embryonic development in mouse. Recently, its human homolog, human mrhl (Hmrhl) was uncovered and study revealed its differential expression in several type of cancers, notably leukemia. In the present study, we further characterize molecular features of lncRNA Hmrhl and gain insight into its functional role in leukemia by gene silencing and transcriptome-based studies. Results indicate its high expression in CML patient samples as well as in K562 cell line. Silencing experiments suggest role of Hmrhl in cell proliferation, migration & invasion in K562 cells. RNA-seq and ChiRP-seq data analysis further revealed its association with important biological processes, including perturbed expression of crucial TFs and cancer-related genes. Among them ZIC1, PDGRFβ and TP53 were identified as regulatory targets, with high possibility of triplex formation by Hmrhl at their promoter site. In addition, we also found TAL-1 to be a potential regulator of Hmrhl expression in K562 cells. Thus, we hypothesize that Hmrhl lncRNA may play a significant role in the pathobiology of CML.
... They have the advantage of determining sequences through amplifications of simple DNA fragments. However, it has limitations such as its high cost, its short reading frames (35 to 500 base pairs) compared to original sequencing (1,000 to 1,200 base pairs), and large amounts of data generation, requiring specialized software (Ansorge, 2009) (Frese et al., 2013). This massively parallel sequencing has a wide variety of applications, including: sequencing of entire genome of a species, non-invasive prenatal testing, identification of genes related to a disease, precise analysis of RNA sequences, identification and analysis of DNA regions that interact with regulatory proteins in gene expression, among others (Buermans et al., 2014) (Ansorge, 2009). ...
Article
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Cancer is a disease that has positioned itself as the second cause of death globally. However, there is a deficiency in diagnostic tools and treatment. Traditionally, biopsy involves tissues extraction to be examined. But, it is an invasive procedure and can lead to complications in patients. Liquid biopsy has been developed as a non-invasive technique, with lower cost and allowing a better diagnosis. As a complement, new-generation sequencing (NGS) allows information sequencing and analysis from DNA. The combined use of these tools can result in identification of genetic aberrations in cancer, improving of patients´ prognosis, and use of personalized therapies when studying an individual´s genomics, epigenomics, and proteomics profile. Despite these advances, there are elements to improve procedures sensitivity, a better understanding of biochemistry and immunological response in tumorigenesis processes, and integration of this data through computational strategies for the development of tumor profiles.
... Recently, several groups have evaluated new methods to assess MSI using next generation sequencing (NGS) technologies from tumor and/or normal tissue pairs (13)(14)(15). NGS refers to a group of technologies which have, in common, the ability to perform and capture data from millions of sequencing reactions simultaneously, also called massively parallel sequencing (16). Hause et al. developed the MOSAIC meth-od for crosssectional MSI analysis in 18 cancer types including CRC using the cancer exomes from the Cancer Genome Atlas database. ...
Article
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Microsatellites are short tandem repeat DNA sequences of one to tetra base pairs distributed throughout the human genome, both in coding and non-coding regions. Owing to their repeated structure, microsatellites are particularly prone to replication errors that are normally repaired by the Mismatch Repair (MMR) system. MMR is a very highly conserved cellular process, involving many proteins, resulting in the identification, and subsequent repair of mismatched bases, likely to have arisen during DNA replication, genetic recombination or chemical or physical damage. Proteins within the MMR system include MLH1, PMS2, MSH2, MSH6, MLH3, MSH3, PMS1, and Exo1. Deficient MMR (dMMR) results in a strong mutator phenotype known as microsatellite instability (MSI), characterized by widespread length polymor-phisms of microsatellite sequences due to DNA polymerase slippage. MSI is recognized as one of the major carcinogenetic pathways of colorectal cancer (CRC): it represents a molecular hallmark of hereditary non-polyposis colorectal cancer (HNPCC), also known as Lynch syndrome (LS); moreover it is detected in 15% of sporadic colorectal cancers, more often due to an epigenetic inactivation of MLH1. Identification of MSI CRC is important, as MSI may serve as a screening tool for detecting LS, a prognostic marker for patient outcome , and a predictive marker for response to chemotherapy and to immunotherapy. (www.actabiomedica.it)