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Montmayeur, Anna M., Terry Fei Fan Ng, Alexander Schmidt, Kun Zhao, Laura Magaña, Jane Iber, Christina J. Castro et al. "High-Throughput Next-Generation Sequencing of Polioviruses". Journal of Clinical Microbiology 55, n. 2 (7 dicembre 2016): 606–15. http://dx.doi.org/10.1128/jcm.02121-16.
Testo completoAbstract (sommario):
ABSTRACTThe poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.
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Li, Niannian, Kairang Jin, Yanmin Bai, Haifeng Fu, Lin Liu e Bin Liu. "Tn5 Transposase Applied in Genomics Research". International Journal of Molecular Sciences 21, n. 21 (6 novembre 2020): 8329. http://dx.doi.org/10.3390/ijms21218329.
Testo completoAbstract (sommario):
The development of high-throughput sequencing (next-generation sequencing technology (NGS)) and the continuous increase in experimental throughput require the upstream sample processing steps of NGS to be as simple as possible to improve the efficiency of the entire NGS process. The transposition system has fast “cut and paste” and “copy and paste” functions, and has been innovatively applied to the NGS field. For example, the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) uses high-throughput sequencing to detect chromatin regions accessible by Tn5 transposase. Linear Amplification via Transposon Insertion (LIANTI) uses Tn5 transposase for linear amplification, haploid typing, and structural variation detection. Not only is it efficient and simple, it effectively shortens the time for NGS sample library construction, realizes large-scale and rapid sequencing, improves sequencing resolution, and can be flexibly modified for more technological innovation.
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Suravajhala, Prashanth, e Alexey Goltsov. "Three Grand Challenges in High Throughput Omics Technologies". Biomolecules 12, n. 9 (4 settembre 2022): 1238. http://dx.doi.org/10.3390/biom12091238.
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Saeed, Muhammad, Zainab Jamil, Tayyab Shehzad, Syed Zia ul Hasan, Riffat Bibi, Safia Naureen Malik, Hafiz Matee-ur-Rehman e Raees Ahmed. "Role of Next Generation Sequencing (NGS) in Plant Disease Management: A Review". Journal of Applied Research in Plant Sciences 4, n. 01 (23 febbraio 2023): 512–17. http://dx.doi.org/10.38211/joarps.2023.04.01.61.
Testo completoAbstract (sommario):
A high throughput technique used to determine a part of the nucleotide sequence of an organism’s genome is called next generation sequencing (NGS). NGS has been Proven revolutionary in genomics. Clinical diagnostics, Plant diseases diagnostic and other aspects of medical are now made possible by sequencing. Techniques of NGS: there are different techniques of NGS which are being used in real life sciences i.e., Illumina sequencing, Pyrosequencing, Roche 454 sequencing and Ion torrent sequencing. All vintage methods like culturing in bacterial, fungal, and viral samples are being suppressed by next generation sequencing. The potential for random metagenomic sequencing of sick samples to find potential pathogens has surfaced with the development of next-generation high-throughput parallel sequencing technology. NGS enables highly efficient, rapid, low-cost DNA or RNA high-throughput sequencing of plant virus and viroids genomes, as well as specific small RNAs generated during infection. Although this technique is not so much familiar in the field of plant diseases. However, its widespread application in agronomic sciences will make it possible to create solutions to future food-related challenges that involve biotic stress.
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Williams, Gareth Haydn, Robert Paul Thatcher, Tiffany Eira Haddow, Keeda-Marie Hardisty e Marco Loddo. "Immunofocus-PD-L1 high throughput quantitative next generation sequencing assay." Journal of Clinical Oncology 38, n. 15_suppl (20 maggio 2020): e13521-e13521. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e13521.
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e13521 Background: Immunohistochemical (IHC) assays are presently used as the gold standard predictive tests for immunotherapy but are compromised due to a number of potential variables. Comparative studies have demonstrated differing levels of PD-L1 staining between assays which appears independent of the antibody binding epitope. Secondly, inter-reader reliability even between expert pathologists is problematic particularly for assessment of PD-L1 positive immune cell populations. Methods: To improve predictive testing for anti PD-L1/PD1 immunotherapies we have developed and validated a Next Generation Sequencing Platform, Immunofocus, able to perform high-throughput quantitative PD-L1 gene expression levels in routine diagnostic PWET biopsies. We applied Immunofocus to a cohort of 130 NSCLCs and compared PD-L1 gene expression levels with PD-L1 IHC scores generated using the VENTANA PD-L1 (SP142) Assay. The PD-L1 IHC assessment was carried out double blinded by an independent laboratory. PD-L1 IHC scores were calculated using an algorithm combining tumour proportion score (TPS) with a PD-L1 positive immune cell (IC) score and immune cell area. Results: An exceptionally high degree of correlation was observed between the NGS PD-L1 levels with the combined PD-L1 IHC scores (P < 0.001). Therapeutic cut points for NGS PD-L1 levels were identified corresponding to PD-L1 IHC defined clinical cut points. Notably, ~20% of patients with negative PD-L1 IHC scores showed high NGS PD-L1 expression levels. We hypothesize that these cases represent false negatives and identify a cohort of patients who have shown significant response rates to anti-PD-L1/PD-directed immunotherapies. Conclusions: The Immunofocus NGS PD-L1 assay has potential to greatly improve patient selection for immunotherapy by removing the IHC assay variables and inter-reader variability which compromise current PD-L1 IHC tests while also providing standardized high throughput in the clinical setting. Immunofocus is able to integrate gene expression with somatic mutation analysis allowing capture of networks regulating the immune-checkpoint including for example adaptive and innate resistance pathways, JAK1/2 pathways, differential MHC expression, TEFF gene signature, neoantigen surrogates such as DDR defects and TMB. The integration of NGS PD-L1 expression with other putative biomarkers of response is presently ongoing to further improve prediction of response.
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Kechin, Andrey, Viktoria Borobova, Ulyana Boyarskikh, Evgeniy Khrapov, Sergey Subbotin e Maxim Filipenko. "NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions". PLOS Computational Biology 16, n. 12 (30 dicembre 2020): e1008468. http://dx.doi.org/10.1371/journal.pcbi.1008468.
Testo completoAbstract (sommario):
Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes.
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Wang, Qi, Lijuan Cao, Guangying Sheng, Hongjie Shen, Jing Ling, Jundan Xie, Zhenni Ma et al. "Application of High-Throughput Sequencing in the Diagnosis of Inherited Thrombocytopenia". Clinical and Applied Thrombosis/Hemostasis 24, n. 9_suppl (13 agosto 2018): 94S—103S. http://dx.doi.org/10.1177/1076029618790696.
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Inherited thrombocytopenia is a group of hereditary diseases with a reduction in platelet count as the main clinical manifestation. Clinically, there is an urgent need for a convenient and rapid diagnosis method. We introduced a high-throughput, next-generation sequencing (NGS) platform into the routine diagnosis of patients with unexplained thrombocytopenia and analyzed the gene sequencing results to evaluate the value of NGS technology in the screening and diagnosis of inherited thrombocytopenia. From a cohort of 112 patients with thrombocytopenia, we screened 43 patients with hereditary features. For the blood samples of these 43 patients, a gene sequencing platform for hemorrhagic and thrombotic diseases comprising 89 genes was used to perform gene detection using NGS technology. When we combined the screening results with clinical features and other findings, 15 (34.9%) of 43patients were diagnosed with inherited thrombocytopenia. In addition, 19 pathogenic variants, including 8 previously unreported variants, were identified in these patients. Through the use of this detection platform, we expect to establish a more effective diagnostic approach to such disorders.
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Wang, Jia-Wang, Wenxiu Zhang, Yan Zhang, Jiajia Zhou, Jing Li, Min Zhang, Shanshan Wen et al. "Reproducible and high sample throughput isomiR next-generation sequencing for cancer diagnosis." Journal of Clinical Oncology 42, n. 16_suppl (1 giugno 2024): e15013-e15013. http://dx.doi.org/10.1200/jco.2024.42.16_suppl.e15013.
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e15013 Background: Next-generation sequencing (NGS) can produce up to 6 Tb of data per run with single-nucleotide accuracy, making it ideal for quantifying isomiRs, which encompass both canonical miRNAs and their variants, for clinical applications. However, NGS has poor reproducibility and low sample throughput in quantifying circulating isomiRs due to significant technical variations and the limitations of the multiplex strategy, as evidenced by the fact that no isomiR NGS technique has been successfully used to diagnose cancer. Methods: To address these challenges, a library construction method including a dual unique-dual-index (DUDI) technology was developed. DUDI uses a pair of Inner UDI (IUDI) and outer UDI (OUDI) to label a sample. Twelve independent batches of isomiR NGS were carried out, including three repeated batches. Each batch included 100 gastric cancer and 100 control plasma samples. Batch effect, correlation coefficient (R), and principal component (PCA) analyses were used to evaluate technical reproducibility. Machine learning binary classification was used to assess biological reproducibility, with each pair of batch data serving interchangeably as both training and testing data. Results: In this multicenter study, over 700G of isomiR data were generated from 402 gastric cancer and 498 control samples, with a maximum error rate of 1 in 7 million isomiRs being assigned to wrong samples. The PCA plot indicates high technical reproducibility across the three repeated batches, shown by the extensive intermingling of data points from each batch and the lack of distinct batch-wise clustering. This observation is reinforced by that the R value for each of 239 isomiRs between the repeated batches are close to 1. While the mutual machine learning validations between the repeated batches yielded ~95% accuracy, indicating high biological reproducibility. The accuracies of the validations between the different batches of different samples range from 70% to 82%. The lower accuracy is as expected, given the high genetic heterogeneity of cancer and the small sample size. Furthermore, the IsomiR differentiated expression profiles from the current NGS study closely match those from prior qPCR studies. Conclusions: The DUDI library construction method can produce reproducible high sample throughput NGS data, yet it is cost-effective and straightforward. The maximum number of samples that can be multiplexed in an NGS project is almost one million, i.e., 976 * 976, as IUDI and OUDI can be any of the 976 designed DUDIs. This number far exceeds the high sample throughput requirements of any NGS application. While the capability to distinguish true biological variations of IsomiRs from technical noise, demonstrated by the high technical and biological reproducibility and concordance with the qPCR data, enables the development of robust machine learning algorithms for cancer diagnostics.
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Tripathi, Pooja, Jyotsna Singh, Jonathan A. Lal e Vijay Tripathi. "Next-Generation Sequencing: An Emerging Tool for Drug Designing". Current Pharmaceutical Design 25, n. 31 (14 novembre 2019): 3350–57. http://dx.doi.org/10.2174/1381612825666190911155508.
Testo completoAbstract (sommario):
Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.
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Chae, H., S. Rhee, K. P. Nephew e S. Kim. "BioVLAB-MMIA-NGS: microRNA-mRNA integrated analysis using high-throughput sequencing data". Bioinformatics 31, n. 2 (29 settembre 2014): 265–67. http://dx.doi.org/10.1093/bioinformatics/btu614.
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Beniwal, Nisha, e Baljeet Singh Saharan. "Emerging Importance of Viral Transport Media in High-Throughput Sequencing Fidelity for Genomic Analysis". Journal of Scientific Research and Reports 29, n. 10 (7 novembre 2023): 99–103. http://dx.doi.org/10.9734/jsrr/2023/v29i101801.
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The impact of Viral Transport Media (VTM) components on the efficacy and accuracy of high-throughput Next-Generation Sequencing (NGS) techniques is a subject of paramount importance in the field of viral genomics and diagnostics. VTMs play a pivotal role in preserving viral samples, and their composition can profoundly influence the quality and reliability of NGS results. This study explores the intricate relationship between VTM components and NGS outcomes, with a focus on optimizing diagnostic precision and the efficiency of NGS in viral genomics. Through comprehensive investigation, this research sheds light on the critical implications of VTM composition for enhancing viral genomic analysis, streamlining diagnostic processes, and bolstering public health responses. The findings have far-reaching consequences, including the potential for personalized medicine, rapid detection of emerging viral threats, and the improvement of public health surveillance. Standardized protocols and ongoing technological advancements are essential in harnessing the full potential of NGS for genomics and metagenomics, marking a significant step toward more effective viral disease management and outbreak response.
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Gerilovych, A. P., M. I. Sushko, S. S. Mandyhra, N. S. Rodyna, M. Ye Romanko, M. V. Kuchinskiy e I. O. Gerilovych. "APPLICATION OF THE NEXT GENERATION SEQUENCING IN BIOLOGY AND MEDICINE". One Health Journal 2, n. I (6 marzo 2024): 32–44. http://dx.doi.org/10.31073/onehealthjournal2024-i-05.
Testo completoAbstract (sommario):
Next-Generation Sequencing (NGS), also known as high-throughput sequencing, refers to a set of modern DNA sequencing technologies that have revolutionized the field of genomics. Advantages of NGS techniques involving high speed (parallel sequencing is faster than traditional methods, allowing researchers to obtain results more quickly), cost-effectiveness (ability to sequence multiple fragments simultaneously reduces the cost per base compared to traditional sequencing), and scalability (platforms can be scaled to accommodate varying levels of throughput depending on experimental needs). NGS has significantly accelerated genomics research, enabling breakthroughs in fields such as personalized medicine, cancer genomics, and evolutionary biology. However, challenges such as data analysis complexity, error rates, and cost still exist and are areas of ongoing research and improvement within the field of sequencing technologies. Paper contains the brief explanation of the current NGS platforms and their features. NGS biomedical application is described with the main advantages and abilities of the analysed tools.
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Ariyadasa, Ruvini, e Nils Stein. "Advances in BAC-Based Physical Mapping and Map Integration Strategies in Plants". Journal of Biomedicine and Biotechnology 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/184854.
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In the advent of next-generation sequencing (NGS) platforms, map-based sequencing strategy has been recently suppressed being too expensive and laborious. The detailed studies on NGS drafts alone indicated these assemblies remain far from gold standard reference quality, especially when applied on complex genomes. In this context the conventional BAC-based physical mapping has been identified as an important intermediate layer in current hybrid sequencing strategy. BAC-based physical map construction and its integration with high-density genetic maps have benefited from NGS and high-throughput array platforms. This paper addresses the current advancements of BAC-based physical mapping and high-throughput map integration strategies to obtain densely anchored well-ordered physical maps. The resulted maps are of immediate utility while providing a template to harness the maximum benefits of the current NGS platforms.
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Salado, Isabel, Alberto Fernández-Gil, Carles Vilà e Jennifer A. Leonard. "Automated genotyping of microsatellite loci from feces with high throughput sequences". PLOS ONE 16, n. 10 (25 ottobre 2021): e0258906. http://dx.doi.org/10.1371/journal.pone.0258906.
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Ecological and conservation genetic studies often use noninvasive sampling, especially with elusive or endangered species. Because microsatellites are generally short in length, they can be amplified from low quality samples such as feces. Microsatellites are highly polymorphic so few markers are enough for reliable individual identification, kinship determination, or population characterization. However, the genotyping process from feces is expensive and time consuming. Given next-generation sequencing (NGS) and recent software developments, automated microsatellite genotyping from NGS data may now be possible. These software packages infer the genotypes directly from sequence reads, increasing throughput. Here we evaluate the performance of four software packages to genotype microsatellite loci from Iberian wolf (Canis lupus) feces using NGS. We initially combined 46 markers in a single multiplex reaction for the first time, of which 19 were included in the final analyses. Megasat was the software that provided genotypes with fewer errors. Coverage over 100X provided little additional information, but a relatively high number of PCR replicates were necessary to obtain a high quality genotype from highly unoptimized, multiplexed reactions (10 replicates for 18 of the 19 loci analyzed here). This could be reduced through optimization. The use of new bioinformatic tools and next-generation sequencing data to genotype these highly informative markers may increase throughput at a reasonable cost and with a smaller amount of laboratory work. Thus, high throughput sequencing approaches could facilitate the use of microsatellites with fecal DNA to address ecological and conservation questions.
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Ishiya, Koji, e Shintaroh Ueda. "MitoSuite: a graphical tool for human mitochondrial genome profiling in massive parallel sequencing". PeerJ 5 (30 maggio 2017): e3406. http://dx.doi.org/10.7717/peerj.3406.
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Recent rapid advances in high-throughput, next-generation sequencing (NGS) technologies have promoted mitochondrial genome studies in the fields of human evolution, medical genetics, and forensic casework. However, scientists unfamiliar with computer programming often find it difficult to handle the massive volumes of data that are generated by NGS. To address this limitation, we developed MitoSuite, a user-friendly graphical tool for analysis of data from high-throughput sequencing of the human mitochondrial genome. MitoSuite generates a visual report on NGS data with simple mouse operations. Moreover, it analyzes high-coverage sequencing data but runs on a stand-alone computer, without the need for file upload. Therefore, MitoSuite offers outstanding usability for handling massive NGS data, and is ideal for evolutionary, clinical, and forensic studies on the human mitochondrial genome variations. It is freely available for download from the website https://mitosuite.com.
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Zheng, Weibo, Jing Chen, Thomas G. Doak, Weibo Song e Ying Yan. "ADFinder: accurate detection of programmed DNA elimination using NGS high-throughput sequencing data". Bioinformatics 36, n. 12 (4 aprile 2020): 3632–36. http://dx.doi.org/10.1093/bioinformatics/btaa226.
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Abstract Motivation Programmed DNA elimination (PDE) plays a crucial role in the transitions between germline and somatic genomes in diverse organisms ranging from unicellular ciliates to multicellular nematodes. However, software specific for the detection of DNA splicing events is scarce. In this paper, we describe Accurate Deletion Finder (ADFinder), an efficient detector of PDEs using high-throughput sequencing data. ADFinder can predict PDEs with relatively low sequencing coverage, detect multiple alternative splicing forms in the same genomic location and calculate the frequency for each splicing event. This software will facilitate research of PDEs and all down-stream analyses. Results By analyzing genome-wide DNA splicing events in two micronuclear genomes of Oxytricha trifallax and Tetrahymena thermophila, we prove that ADFinder is effective in predicting large scale PDEs. Availability and implementation The source codes and manual of ADFinder are available in our GitHub website: https://github.com/weibozheng/ADFinder. Supplementary information Supplementary data are available at Bioinformatics online.
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Zubov, V. V., D. A. Chemeris, R. G. Vasilov, V. E. Kurochkin e Ya I. Alekseev. "Brief history of high-throughput nucleic acid sequencing methods." Biomics 13, n. 1 (2021): 27–46. http://dx.doi.org/10.31301/2221-6197.bmcs.2021-4.
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The processes occurring during the enzymatic growth of the DNA chain in the form of elongation of the molecules, the release of pyrophosphate, proton, thermal energy, and an increase in electrical impedance, which are used in various methods of high-throughput DNA sequencing by synthesis, are briefly considered. The detection of DNA chain growth is controlled by high-voltage gel electrophoresis and has limited scalability. As for mentioned above other by-products of DNA chain polymerization, their detection can be easily scalable, which has led to the emergence of methods for whole genome sequencing of new generations of DNA, which have received the widely used abbreviation NGS - Next Generation Sequencing. However, the attribution of any new sequencing method to a particular generation is sometimes difficult due to the fact that the principle used in it was born earlier than the other one was implemented, which turned out to be less productive in the end. In addition, it is more important to distinguish the methods of new DNA sequencing into two groups in which the massive parallel sequencing of identical matrices takes place or the sequencing of single DNA molecules takes place and last one have received the designation monomolecular sequencing. In this review, along with the classical Sanger method of DNA sequencing, which is still the "gold standard", pyrosequencing, semiconductor sequencing, thermosequencing, electronic sequencing, fluorescent bridge sequencing and sequencing using nanoballs from the first group, as well as monomolecular methods – tSMS sequencing, SMRT sequencing and nanopore sequencing are considered. Attention is paid to the costs of DNA sequencing and the prospects for its development.
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Bieler, Jonathan, Christian Pozzorini, Jessica Garcia, Alex C. Tuck, Morgane Macheret, Adrian Willig, Sébastien Couraud et al. "High-Throughput Nucleotide Resolution Predictions of Assay Limitations Increase the Reliability and Concordance of Clinical Tests". JCO Clinical Cancer Informatics, n. 5 (ottobre 2021): 1085–95. http://dx.doi.org/10.1200/cci.21.00057.
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PURPOSE The ability of next-generation sequencing (NGS) assays to interrogate thousands of genomic loci has revolutionized genetic testing. However, translation to the clinic is impeded by false-negative results that pose a risk to patients. In response, regulatory bodies are calling for reliability measures to be reported alongside NGS results. Existing methods to estimate reliability do not account for sample- and position-specific variability, which can be significant. Here, we report an approach that computes reliability metrics for every genomic position and sample interrogated by an NGS assay. METHODS Our approach predicts the limit of detection (LOD), the lowest reliably detectable variant fraction, by taking technical factors into account. We initially explored how LOD is affected by input material amount, library conversion rate, sequencing coverage, and sequencing error rate. This revealed that LOD depends heavily on genomic context and sample properties. Using these insights, we developed a computational approach to predict LOD on the basis of a biophysical model of the NGS workflow. We focused on targeted assays for cell-free DNA, but, in principle, this approach applies to any NGS assay. RESULTS We validated our approach by showing that it accurately predicts LOD and distinguishes reliable from unreliable results when screening 580 lung cancer samples for actionable mutations. Compared with a standard variant calling workflow, our approach avoided most false negatives and improved interassay concordance from 94% to 99%. CONCLUSION Our approach, which we name LAVA (LOD-aware variant analysis), reports the LOD for every position and sample interrogated by an NGS assay. This enables reliable results to be identified and improves the transparency and safety of genetic tests.
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Nellimarla, Srinivas, e Prasad Kesanakurti. "Next-Generation Sequencing: A Promising Tool for Vaccines and Other Biological Products". Vaccines 11, n. 3 (23 febbraio 2023): 527. http://dx.doi.org/10.3390/vaccines11030527.
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Next-generation sequencing (NGS), also known as high-throughput sequencing (HTS), is a commonly used term to represent a set of DNA sequencing technologies that have been in use for almost two decades [...]
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Javitt, Gail H., e Katherine Strong Carner. "Regulation of Next Generation Sequencing". Journal of Law, Medicine & Ethics 42, S1 (2014): 9–21. http://dx.doi.org/10.1111/jlme.12159.
Testo completoAbstract (sommario):
Since the first draft of the human genome was published in 2001, DNA sequencing technology has advanced at a remarkable pace. Launched in 1990, the Human Genome Project sought to sequence all three billion base pairs of the haploid human genome, an endeavor that took more than a decade and cost nearly three billion dollars. The subsequent development of so-called “next generation” sequencing (NGS) methods has raised the possibility that real-time, affordable genome sequencing will soon be widely available. Currently, NGS methods can be used to sequence up to 60 billion base pairs per day. Whole-genome sequencing costs an estimated $5,000-10,000, with that number predicted to fall to $1000 in the near future.In the past few years, the availability of high-throughput NGS methods has led to a proliferation of potential and actual clinical applications for NGS. NGS therefore has the potential to usher in the long-awaited era of personalized medicine.
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Roy, Denis, Sarah J. Lehnert, Clare J. Venney, Ryan Walter e Daniel D. Heath. "NGS-μsat: bioinformatics framework supporting high throughput microsatellite genotyping from next generation sequencing platforms". Conservation Genetics Resources 13, n. 2 (11 gennaio 2021): 161–73. http://dx.doi.org/10.1007/s12686-020-01186-0.
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Wagner, Ines, Daniel Schefzyk, Jens Pruschke, Gerhard Schoefl, Bianca Schoene, Kathrin Lang, Jan A. Hofmann et al. "OR28 High-throughput KIR sequencing by NGS: 500,000 registry samples genotyped at allelic resolution". Human Immunology 78 (settembre 2017): 27. http://dx.doi.org/10.1016/j.humimm.2017.06.034.
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Nelsen, Donald J., Rohita Sinha, Aaron J. Tyler, Jordyn Westergaard, Jamie Nutt, Mark Wissel, Steve Kleiboeker e Michelle Altrich. "268. Fungal NGS: Identification of Etiological Agents of Invasive Fungal Infection by High-throughput Sequencing". Open Forum Infectious Diseases 6, Supplement_2 (ottobre 2019): S148—S149. http://dx.doi.org/10.1093/ofid/ofz360.343.
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Abstract Background Invasive fungal infections (IFI) cause severe symptoms that affect immunocompromised and transplant patient populations. Antifungal therapies vary depending on the pathogenic species, and delays in diagnosis can lead to graft loss and an increase in morbidity and mortality. Therefore, rapid identification of fungi causing IFI is critical for informing antifungal therapy. Such actionable genus/species information can be obtained quickly via Next-generation Sequencing (NGS). In this study, an NGS assay was developed to identify fungal species responsible for IFI, allowing for selection of effective antifungal therapies. Methods Internal transcribed spacer (ITS) regions 1 and 2 were used for fungal identification. Primers were taken from published research and/or designed/modified by assessment in fungal sequence alignments. A DNA sequence database was compiled and a reference-assisted assembly approach utilizing % sequence ID and % coverage was developed for species identification. End-point PCR was conducted on DNA extracted from 19 pathogenic fungal species, and mixed communities (MC) for preliminary sensitivity and inclusivity. Sensitivity was assessed using dilutions of template DNA into the PCR reaction. Results NGS data of 14 individual species and 4 MC passed quality control checks. Using only ITS1 and ITS2, species identification was expected for 10 of 14 individuals. We observed species identification in 9 individual samples, and 13 were identified within the top 5 results. All individuals were identified to genus. In MC analyses, combinations of 3, 4, 6, and 10 fungal species resolved 100% of the genera present, but failed to resolve species adequately with only 2 loci evaluated. Unexpectedly, 3 tested Aspergillus spp. were correctly identified with this limited data in both single and MC samples. The lower limit of detection was assessed at 5,000 genomic equivalents/mL of eluate. Conclusion The inclusivity and sensitivity demonstrated here of an NGS approach for identification of etiological agents of IFI support this assay’s potential utility as an aid in the treatment of IFI in at-risk patient groups. This assay allows for rapid identification (<4 days) of fungal species to aid clinicians in improving case outcomes. Disclosures All authors: No reported disclosures.
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Colabella, Claudia, Debora Casagrande Pierantoni, Laura Corte, Luca Roscini, Angela Conti, Matteo Bassetti, Carlo Tascini, Vincent Robert e Gianluigi Cardinali. "Single Strain High-Depth NGS Reveals High rDNA (ITS-LSU) Variability in the Four Prevalent Pathogenic Species of the Genus Candida". Microorganisms 9, n. 2 (2 febbraio 2021): 302. http://dx.doi.org/10.3390/microorganisms9020302.
Testo completoAbstract (sommario):
Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology.
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Hassouneh, Ramzi. "Is Next-Generation Sequencing Appropriate for the Clinic?" University of Ottawa Journal of Medicine 4, n. 2 (17 novembre 2014): 45–48. http://dx.doi.org/10.18192/uojm.v4i2.1075.
Testo completoAbstract (sommario):
Next-generation sequencing (NGS) has ignited a revolution in genomic medicine by eliminating the inherent limitations of conventional sequencing methods. Due to its high throughput and low-cost, clinics can use NGS to perform targeted and genome sequencing to make diagnoses or pre-screen for risk to future disease. Despite its clinical uses, many challenges exist before NGS becomes a mainstay in the clinic. There is a lack of understanding of the impact of genetic variants on health and disease and how to best apply genetic information to patient care. Nevertheless, the translation of base pair reads to clinical applications has truly begun.
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Liaudanski, A. D., R. S. Shulinski, Y. A. Mishuk e L. N. Sivitskaya. "COMPARISON OF GENOTYPE PHASING METODS FOR THE HIGH THROUGHPUT SEQUENCING DATA OF CLINICAL EXOMES". Молекулярная и прикладная генетика 31 (8 dicembre 2021): 114–23. http://dx.doi.org/10.47612/1999-9127-2021-31-114-123.
Testo completoAbstract (sommario):
The aim of this investigation was to search for an algorithm for phasing of exome NGS data that would be optimal for analyzing the cis-/transposition of closely located polymorphic loci in patients with hereditary diseases. The NGS data on clinical exome sequences from 149 patients was collected and analyzed, and its primary processing was carried out. A combined reference panel for phasing and genotype imputation “Belref1000G” was created by adding 131 samples of Belarusian patients to the panel from the “1000 Genomes” project. The most effective methods of phasing have been determined: the Michigan Imputation Server (online service) allows achieving the highest accuracy of phasing data from clinical exomes in Belarus. In cases where an online format of phasing is not appropriate for whatever reason, Beagle software with the combined reference panel “Belref1000G” is recommended for use. Beagle software with the “1000G” reference panel should be used to obtain the longest phasing blocks.
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Nowrousian, Minou. "Next-Generation Sequencing Techniques for Eukaryotic Microorganisms: Sequencing-Based Solutions to Biological Problems". Eukaryotic Cell 9, n. 9 (2 luglio 2010): 1300–1310. http://dx.doi.org/10.1128/ec.00123-10.
Testo completoAbstract (sommario):
ABSTRACT Over the past 5 years, large-scale sequencing has been revolutionized by the development of several so-called next-generation sequencing (NGS) technologies. These have drastically increased the number of bases obtained per sequencing run while at the same time decreasing the costs per base. Compared to Sanger sequencing, NGS technologies yield shorter read lengths; however, despite this drawback, they have greatly facilitated genome sequencing, first for prokaryotic genomes and within the last year also for eukaryotic ones. This advance was possible due to a concomitant development of software that allows the de novo assembly of draft genomes from large numbers of short reads. In addition, NGS can be used for metagenomics studies as well as for the detection of sequence variations within individual genomes, e.g., single-nucleotide polymorphisms (SNPs), insertions/deletions (indels), or structural variants. Furthermore, NGS technologies have quickly been adopted for other high-throughput studies that were previously performed mostly by hybridization-based methods like microarrays. This includes the use of NGS for transcriptomics (RNA-seq) or the genome-wide analysis of DNA/protein interactions (ChIP-seq). This review provides an overview of NGS technologies that are currently available and the bioinformatics analyses that are necessary to obtain information from the flood of sequencing data as well as applications of NGS to address biological questions in eukaryotic microorganisms.
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Алексеева, Е. А., О. В. Бабенко, В. М. Козлова, Т. Л. Ушакова, Т. П. Казубская, А. С. Танас, К. И. Карандашева, В. В. Стрельников e Д. В. Залетаев. "Advantages of high throughput parallel sequencing in detecting somatic mosaicism in sporadic retinoblastoma". Nauchno-prakticheskii zhurnal «Medicinskaia genetika», n. 6(215) (29 giugno 2020): 6–7. http://dx.doi.org/10.25557/2073-7998.2020.06.6-7.
Testo completoAbstract (sommario):
Почти 80% случаев наследственной ретинобластомы не имеют семейного анамнеза и возникают в результате мутаций de novo в гене RB1. Методом высокопроизводительного параллельного секвенирования (ВПС) проведено молекулярно-генетическое обследование 208 неродственных больных со спорадической РБ, среди которых 145 пациентов с унилатеральной формой заболевания и 63 - с билатеральной. В группе пациентов с билатеральной РБ молекулярные изменения в гене RB1 обнаружены в 90,5% (57/63) случаев. У 4,8% (3/63) пациентов определен мозаичный вариант мутации в гене RB1. В группе пациентов с унилатеральной РБ молекулярные изменения в гене RB1 выявлены в 17,9% (26/145) случаев. Среди исследованных пациентов соматический мозаицизм выявлен в 9,0% (13/165) случаев. Применение ВПС позволяет точно определять аллельную частоту вариантов, что делает поиск соматического мозаицизма эффективным. Almost 80% of cases of hereditary retinoblastoma do not have a family history and arise as a result of de novo mutations in the RB1 gene. An NGS test was performed on 208 unrelated patients with sporadic RB, including 145 patients with a unilateral form and 63 patients with a bilateral one. In the group of patients with bilateral RB, pathogenic variants in the RB1 gene were detected in 90.5% (57/63) cases. In 4.8% (3/63) of patients, a mosaic variants were determined. In the group of patients with unilateral RB, changes in the RB1 gene were detected in 17.9% (26/145) cases. Among the examined patients, somatic mosaicism was detected in 9.0% (13/165) cases. NGS allows us to determine the allelic frequency of variants, which makes the search for somatic mosaicism effective.
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Park, H., S. Murthy, C. Bott, M. C. M. van Loosdrecht e K. Chandran. "Nationwide metagenome survey of anammox processes via high-throughput next generation sequencing (NGS): 2012-2013". Proceedings of the Water Environment Federation 2014, n. 6 (1 ottobre 2014): 2366–71. http://dx.doi.org/10.2175/193864714815942017.
Testo completo
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Tiu, Charles Kevin, Feng Zhu, Lin-Fa Wang e Ruklanthi de Alwis. "Phage ImmunoPrecipitation Sequencing (PhIP-Seq): The Promise of High Throughput Serology". Pathogens 11, n. 5 (11 maggio 2022): 568. http://dx.doi.org/10.3390/pathogens11050568.
Testo completoAbstract (sommario):
Phage ImmunoPrecipitation Sequencing (PhIP-Seq) is a high throughput serological technology that is revolutionizing the manner in which we track antibody profiles. In this review, we mainly focus on its application to viral infectious diseases. Through the pull-down of patient antibodies using peptide-tile-expressing T7 bacteriophages and detection using next-generation sequencing (NGS), PhIP-Seq allows the determination of antibody repertoires against peptide targets from hundreds of proteins and pathogens. It differs from conventional serological techniques in that PhIP-Seq does not require protein expression and purification. It also allows for the testing of many samples against the whole virome. PhIP-Seq has been successfully applied in many infectious disease investigations concerning seroprevalence, risk factors, time trends, etiology of disease, vaccinology, and emerging pathogens. Despite the inherent limitations of this technology, we foresee the future expansion of PhIP-Seq in both investigative studies and tracking of current, emerging, and novel viruses. Following the review of PhIP-Seq technology, its limitations, and applications, we recommend that PhIP-Seq be integrated into national surveillance programs and be used in conjunction with molecular techniques to support both One Health and pandemic preparedness efforts.
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Beck, Tyler F., James C. Mullikin e Leslie G. Biesecker. "Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants". Clinical Chemistry 62, n. 4 (1 aprile 2016): 647–54. http://dx.doi.org/10.1373/clinchem.2015.249623.
Testo completoAbstract (sommario):
Abstract BACKGROUND Next-generation sequencing (NGS) data are used for both clinical care and clinical research. DNA sequence variants identified using NGS are often returned to patients/participants as part of clinical or research protocols. The current standard of care is to validate NGS variants using Sanger sequencing, which is costly and time-consuming. METHODS We performed a large-scale, systematic evaluation of Sanger-based validation of NGS variants using data from the ClinSeq® project. We first used NGS data from 19 genes in 5 participants, comparing them to high-throughput Sanger sequencing results on the same samples, and found no discrepancies among 234 NGS variants. We then compared NGS variants in 5 genes from 684 participants against data from Sanger sequencing. RESULTS Of over 5800 NGS-derived variants, 19 were not validated by Sanger data. Using newly designed sequencing primers, Sanger sequencing confirmed 17 of the NGS variants, and the remaining 2 variants had low quality scores from exome sequencing. Overall, we measured a validation rate of 99.965% for NGS variants using Sanger sequencing, which was higher than many existing medical tests that do not necessitate orthogonal validation. CONCLUSIONS A single round of Sanger sequencing is more likely to incorrectly refute a true-positive variant from NGS than to correctly identify a false-positive variant from NGS. Validation of NGS-derived variants using Sanger sequencing has limited utility, and best practice standards should not include routine orthogonal Sanger validation of NGS variants.
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Tang, Binhua, Xihan Wang e Victor X. Jin. "COPAR: A ChIP-Seq Optimal Peak Analyzer". BioMed Research International 2017 (2017): 1–4. http://dx.doi.org/10.1155/2017/5346793.
Testo completoAbstract (sommario):
Sequencing data quality and peak alignment efficiency of ChIP-sequencing profiles are directly related to the reliability and reproducibility of NGS experiments. Till now, there is no tool specifically designed for optimal peak alignment estimation and quality-related genomic feature extraction for ChIP-sequencing profiles. We developed open-sourced COPAR, a user-friendly package, to statistically investigate, quantify, and visualize the optimal peak alignment and inherent genomic features using ChIP-seq data from NGS experiments. It provides a versatile perspective for biologists to perform quality-check for high-throughput experiments and optimize their experiment design. The package COPAR can process mapped ChIP-seq read file in BED format and output statistically sound results for multiple high-throughput experiments. Together with three public ChIP-seq data sets verified with the developed package, we have deposited COPAR on GitHub under a GNU GPL license.
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Höing, Ann-Sophie, Alexander E. Volk e Hanno J. Bolz. "Klinische Anwendung der Hochdurchsatz-Sequenzierung (next-generation sequencing, NGS) bei der Diagnostik des Usher-Syndroms". Optometry & Contact Lenses 3, n. 9 (30 ottobre 2023): 308–17. http://dx.doi.org/10.54352/dozv.crqo4102.
Testo completoAbstract (sommario):
Purpose. The aim of this publication is to describe the clinical application of high-throughput sequencing (next-generation sequencing, NGS) and its advantages compared with Sanger sequencing using Usher syndrome as an example. Material and Methods. Genetic diagnostics is subject to rapid progress. The identification of disease-causing genetic alter- ations enables increasingly accurate diagnosis of hereditary diseases, making it possible to give affected individuals more precise information about the nature of the disease (course, treatment options, prognosis). Genetic diagnostics of patients and their relatives enables not only to determine the diagnosis of a disease, but its recurrence risk in the family. Results. Usher syndrome is an autosomal recessively inherited disorder, characterized by sensorineural hearing impairment and vision loss (due to retinitis pigmentosa). Several genes are known to be associated with 3 clinical subtypes of Usher syndrome. Conclusion. Advances in genetic diagnostics, including high-throughput sequencing (next-generation sequencing, NGS), today enable uncomplicated, quick and reliable diagno- sis of genetic eye diseases such as Usher syndrome. Through their own diagnosis (or the diagnosis of their child), patients and families can be informed about the prognosis and use meaningful tools early on. Family members of an affected person can be tested for carriership. Keywords Usher syndrome, next-generation sequencing, genetic testing, genetic counseling, genetic heterogeneity
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Shetty, Omshree, Mamta Gurav, Prachi Bapat, Nupur Karnik, Gauri Wagh, Trupti Pai, Sridhar Epari e Sangeeta Desai. "Moving Next-Generation Sequencing into the Clinic". Indian Journal of Medical and Paediatric Oncology 42, n. 03 (maggio 2021): 221–28. http://dx.doi.org/10.1055/s-0041-1732854.
Testo completoAbstract (sommario):
AbstractWith an advancement in the field of molecular diagnostics, there has been a profound evolution in the testing modalities, especially in the field of oncology. In the past decade, sequencing technology has evolved drastically with the advent of high-throughput next-generation sequencing (NGS). Subsequently, the single-gene tests have been replaced by multigene panel-based assays, deep sequencing, massively parallel whole genome, whole-exome sequencing, and so on. NGS has provided molecular diagnostics professionals a wonderful tool to explore and unearth the genetic alterations, underpinning the pathophysiology of the disease. However, this development has posed new challenges which consist of the following; understanding the technology, types of platforms available, various sequencing strategies, bioinformatics and data analysis algorithm, reporting of various variants, and validation of assays and overall for developing NGS assay for clinical utility. The challenges involved sometimes impede development of these high-end assays in laboratories. The present article provides a broad overview of our journey in setting up the NGS assay in a molecular pathology laboratory at a tertiary care oncology center. We hereby describe various important points and steps to be followed while working on the NGS setup, right from its inception to final drafting of the reports, with inclusion of various validation steps. We aim at providing a beginner’s guide to set up NGS assays in the laboratory using recommended best practices and various international guidelines.
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Vodiasova, E. A., E. S. Chelebieva e O. N. Kuleshova. "The new technologies of high-throughput single-cell RNA sequencing". Vavilov Journal of Genetics and Breeding 23, n. 5 (24 agosto 2019): 508–18. http://dx.doi.org/10.18699/vj19.520.
Testo completoAbstract (sommario):
A wealth of genome and transcriptome data obtained using new generation sequencing (NGS) technologies for whole organisms could not answer many questions in oncology, immunology, physiology, neurobiology, zoology and other fields of science and medicine. Since the cell is the basis for the living of all unicellular and multicellular organisms, it is necessary to study the biological processes at its level. This understanding gave impetus to the development of a new direction – the creation of technologies that allow working with individual cells (single-cell technology). The rapid development of not only instruments, but also various advanced protocols for working with single cells is due to the relevance of these studies in many fields of science and medicine. Studying the features of various stages of ontogenesis, identifying patterns of cell differentiation and subsequent tissue development, conducting genomic and transcriptome analyses in various areas of medicine (especially in demand in immunology and oncology), identifying cell types and states, patterns of biochemical and physiological processes using single cell technologies, allows the comprehensive research to be conducted at a new level. The first RNA-sequencing technologies of individual cell transcriptomes (scRNA-seq) captured no more than one hundred cells at a time, which was insufficient due to the detection of high cell heterogeneity, existence of the minor cell types (which were not detected by morphology) and complex regulatory pathways. The unique techniques for isolating, capturing and sequencing transcripts of tens of thousands of cells at a time are evolving now. However, new technologies have certain differences both at the sample preparation stage and during the bioinformatics analysis. In the paper we consider the most effective methods of multiple parallel scRNA-seq using the example of 10XGenomics, as well as the specifics of such an experiment, further bioinformatics analysis of the data, future outlook and applications of new high-performance technologies.
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Barry, Simon, Yazan Hani Mustafa, Carol McGibney, Laura Royo, Martin Higgins, Wail Mohammed, Mahzar Iqbal et al. "Impact of next generation sequencing in high grade glioma management." Journal of Clinical Oncology 42, n. 16_suppl (1 giugno 2024): e14031-e14031. http://dx.doi.org/10.1200/jco.2024.42.16_suppl.e14031.
Testo completoAbstract (sommario):
e14031 Background: Recent advances in genomic profiling have improved our understanding of the molecular pathogenesis of high grade gliomas. Increased availability of high-throughput Next Generation Sequencing (NGS) allows the identification of genetic alterations which could direct personalized cancer treatments for HGG patients. Methods: All patients with high grade glioma (WHO Grade ≥3 glioma by 2016 or 2022 classification) in our institution, who underwent NGS testing between 2019-2023, were identified from a prospectively maintained electronic database. NGS results, from the Oncomine Precision Assay, a multi-gene panel which tests for 50 different mutations, were collated. Medical records were systematically reviewed to collect demographic, treatment and survival data. Alterations detected on NGS were characterized using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Results: 53 patient samples underwent NGS testing (34 males [64%]; 19 females [36%]). Median age at diagnosis was 52 years (range: 20-72 years). Thirty patients (57%) had an ECOG PS 1 at first clinical review. 18 tumors (34%) were found to have MGMT promoter hypermethylation. 28 patients (53%) were found to have potentially actionable alterations according to the ESCAT scale; 19 (35%) EGFR mutations (ESCAT IIIA level evidence) were detected, 5 PIK3CA (9%) mutations (ESCAT IIIA), 3 FGFR3 (6%) alterations (ESCAT IIB), 2 patients with CDK 4 (4%) copy number gain (ESCAT IIIA), 2 PTPRZ1-MET (4%) fusions (ESCAT IIIA) and 1 BRAF V600E (2%) mutation (ESCAT IB). 4 patients were found to have multiple mutations on NGS. 1 sample had insufficient tissue for analysis. 2 patients (4%) were considered for targeted therapy based on NGS results (1 BRAF V600E mutation and 1 FGFR3 alteration) but both deteriorated in advance of commencing treatment. One patient received nivolumab for MSI-H recurrent GBM (ESCAT IIIB) but progressed within 3 months of commencing treatment. Conclusions: Despite advances in our understanding of the pathogenesis of these high grade gliomas and improved awareness of molecular targets, the delivery of targeted therapy to patients with HGG remains challenging. Rapid access to targeted therapy via compassionate access programmes and improved availability and access to clinical trials for patients with rare cancers and molecular alterations, is paramount.
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Янова, Т. И., И. В. Канивец, С. А. Коростелев, Д. В. Пьянков, В. Ю. Удалова, К. В. Горгишели e Ю. К. Киевская. "NGS for prenatal diagnosis of fetal anomalies". Nauchno-prakticheskii zhurnal «Medicinskaia genetika», n. 11(220) (30 novembre 2020): 65–66. http://dx.doi.org/10.25557/2073-7998.2020.11.65-66.
Testo completoAbstract (sommario):
Использование высокопроизводительного секвенирования в пренатальной диагностике позволило значительно увеличить выявляемость причин аномалий развития плода, определенных при УЗИ. Установление релевантного варианта является важным для постановки диагноза и оценки прогноза. Цель настоящей работы - определить распространенность и структуру моногенных заболеваний, являющихся причиной пороков развития плода при использовании секвенирования нового поколения (NGS). В нашем исследовании было проанализировано 60 образцов ДНК плодов, аномалии развития которых были выявлены при УЗИ во время беременности. Патогенные варианты, являющиеся причиной аномалий развития были найдены у 71% плодов. The use of high-throughput sequencing in prenatal diagnostics has significantly increased the detection of the causes of fetal abnormalities identified by ultrasound. Establishing a relevant option is important for making a diagnosis and evaluating the prognosis. The purpose of this work is to determine the prevalence and structure of monogenic diseases that cause fetal malformations using next generation sequencing (NGS). In our study, we analyzed 60 samples of fetal DNA whose abnormalities were detected by ultrasound during pregnancy. Pathogenic variants were found in 71% of fetuses.
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Satam, Heena, Kandarp Joshi, Upasana Mangrolia, Sanober Waghoo, Gulnaz Zaidi, Shravani Rawool, Ritesh P. Thakare et al. "Next-Generation Sequencing Technology: Current Trends and Advancements". Biology 12, n. 7 (13 luglio 2023): 997. http://dx.doi.org/10.3390/biology12070997.
Testo completoAbstract (sommario):
The advent of next-generation sequencing (NGS) has brought about a paradigm shift in genomics research, offering unparalleled capabilities for analyzing DNA and RNA molecules in a high-throughput and cost-effective manner. This transformative technology has swiftly propelled genomics advancements across diverse domains. NGS allows for the rapid sequencing of millions of DNA fragments simultaneously, providing comprehensive insights into genome structure, genetic variations, gene expression profiles, and epigenetic modifications. The versatility of NGS platforms has expanded the scope of genomics research, facilitating studies on rare genetic diseases, cancer genomics, microbiome analysis, infectious diseases, and population genetics. Moreover, NGS has enabled the development of targeted therapies, precision medicine approaches, and improved diagnostic methods. This review provides an insightful overview of the current trends and recent advancements in NGS technology, highlighting its potential impact on diverse areas of genomic research. Moreover, the review delves into the challenges encountered and future directions of NGS technology, including endeavors to enhance the accuracy and sensitivity of sequencing data, the development of novel algorithms for data analysis, and the pursuit of more efficient, scalable, and cost-effective solutions that lie ahead.
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Cohen-Aharonov, Lyora A., Annie Rebibo-Sabbah, Adar Yaacov, Roy Z. Granit, Merav Strauss, Raul Colodner, Ori Cheshin, Shai Rosenberg e Ronen Eavri. "High throughput SARS-CoV-2 variant analysis using molecular barcodes coupled with next generation sequencing". PLOS ONE 17, n. 6 (21 giugno 2022): e0253404. http://dx.doi.org/10.1371/journal.pone.0253404.
Testo completoAbstract (sommario):
The identification of SARS-CoV-2 variants across the globe and their implications on the outspread of the pandemic, infection potential and resistance to vaccination, requires modification of the current diagnostic methods to map out viral mutations rapidly and reliably. Here, we demonstrate that integrating DNA barcoding technology, sample pooling and Next Generation Sequencing (NGS) provide an applicable solution for large-population viral screening combined with specific variant analysis. Our solution allows high throughput testing by barcoding each sample, followed by pooling of test samples using a multi-step procedure. First, patient-specific barcodes are added to the primers used in a one-step RT-PCR reaction, amplifying three different viral genes and one human housekeeping gene (as internal control). Then, samples are pooled, purified and finally, the generated sequences are read using an Illumina NGS system to identify the positive samples with a sensitivity of 82.5% and a specificity of 97.3%. Using this solution, we were able to identify six known and one unknown SARS-CoV-2 variants in a screen of 960 samples out of which 258 (27%) were positive for the virus. Thus, our diagnostic solution integrates the benefits of large population and epidemiological screening together with sensitive and specific identification of positive samples including variant analysis at a single nucleotide resolution.
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Chen, Jiajia, Daqing Zhang, Wenying Yan, Dongrong Yang e Bairong Shen. "Translational Bioinformatics for Diagnostic and Prognostic Prediction of Prostate Cancer in the Next-Generation Sequencing Era". BioMed Research International 2013 (2013): 1–13. http://dx.doi.org/10.1155/2013/901578.
Testo completoAbstract (sommario):
The discovery of prostate cancer biomarkers has been boosted by the advent of next-generation sequencing (NGS) technologies. Nevertheless, many challenges still exist in exploiting the flood of sequence data and translating them into routine diagnostics and prognosis of prostate cancer. Here we review the recent developments in prostate cancer biomarkers by high throughput sequencing technologies. We highlight some fundamental issues of translational bioinformatics and the potential use of cloud computing in NGS data processing for the improvement of prostate cancer treatment.
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Gupta, Piyush Kumar, Rama Shanker Verma, Maria Frolova e Arkady Ayzenshtadt. "HIGH-THROUGHPUT SEQUENCING ANALYSIS OF MICROBIAL POPULATIONS IN ARCTIC ROCK SAMPLE". SWS Journal of EARTH AND PLANETARY SCIENCES 1, n. 2 (1 ottobre 2019): 29–38. http://dx.doi.org/10.35603/eps2019/issue2.03.
Testo completoAbstract (sommario):
Recent developments in the existing molecular genetic tools have augmented our current understanding towards the deleterious effects of nanoparticles on human health. During building construction, the rapid generation of nanoparticles has greatly affected to human with severe toxicity by generating free radicals inside their body as potential health hazards. However, there is still need of analyzing nanoparticle toxicity based on the type of microbial diversity present on surface and its potential impacts on human health. In this study, we used rocks as raw material collected from Arkhangelsk (arctic) region of Russia and fabricated into particles of nanometer range in size by planetary ball milling. The paper presents data of the elemental composition on the basis of which the value of the specific mass energy of atomization of the raw material of the rock was calculated. The energy parameters of the micro- and nanosystems of the sample were calculated: free surface energy and surface activity.
These nanoparticles were showing minimal cytotoxicity to human embryonic kidney cells in a dose-dependent manner. The high-throughput next-generation sequencing (NGS) was used to perform 16S rRNA metagenomic study for determining the type of microbial diversity present on nanoparticle’s surface. The first highest abundance was found for actinobacteria at phylum taxonomic level indicating a population of gram + ve bacteria having economic importance to human. The second highest abundance was seen for proteobacteria at similar taxonomic level exhibiting population of gram - ve bacteria causing pathogenicity in human. The highest abundance of top 25 microbial species was also discussed in this study. In future, this metagenomic study will also identify other microbial species based on 18S rRNA sequencing.
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Correia, Damien, Olivia Doppelt-Azeroual, Jean-Baptiste Denis, Mathias Vandenbogaert e Valérie Caro. "MetaGenSense : A web application for analysis and visualization of high throughput sequencing metagenomic data". F1000Research 4 (2 aprile 2015): 86. http://dx.doi.org/10.12688/f1000research.6139.1.
Testo completoAbstract (sommario):
The detection and characterization of emerging infectious agents has been a continuing public health concern. High Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies have proven to be promising approaches for efficient and unbiased detection of pathogens in complex biological samples, providing access to comprehensive analyses. As NGS approaches typically yield millions of putatively representative reads per sample, efficient data management and visualization resources have become mandatory. Most usually, those resources are implemented through a dedicated Laboratory Information Management System (LIMS), solely to provide perspective regarding the available information. We developed an easily deployable web-interface, facilitating management and bioinformatics analysis of metagenomics data-samples. It was engineered to run associated and dedicated Galaxy workflows for the detection and eventually classification of pathogens. The web application allows easy interaction with existing Galaxy metagenomic workflows, facilitates the organization, exploration and aggregation of the most relevant sample-specific sequences among millions of genomic sequences, allowing them to determine their relative abundance, and associate them to the most closely related organism or pathogen. The user-friendly Django-Based interface, associates the users’ input data and its metadata through a bio-IT provided set of resources (a Galaxy instance, and both sufficient storage and grid computing power). Galaxy is used to handle and analyze the user’s input data from loading, indexing, mapping, assembly and DB-searches. Interaction between our application and Galaxy is ensured by the BioBlend library, which gives API-based access to Galaxy’s main features. Metadata about samples, runs, as well as the workflow results are stored in the LIMS. For metagenomic classification and exploration purposes, we show, as a proof of concept, that integration of intuitive exploratory tools, like Krona for representation of taxonomic classification, can be achieved very easily. In the trend of Galaxy, the interface enables the sharing of scientific results to fellow team members.
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Correia, Damien, Olivia Doppelt-Azeroual, Jean-Baptiste Denis, Mathias Vandenbogaert e Valérie Caro. "MetaGenSense: A web-application for analysis and exploration of high throughput sequencing metagenomic data". F1000Research 4 (22 agosto 2016): 86. http://dx.doi.org/10.12688/f1000research.6139.2.
Testo completoAbstract (sommario):
The detection and characterization of emerging infectious agents has been a continuing public health concern. High Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies have proven to be promising approaches for efficient and unbiased detection of pathogens in complex biological samples, providing access to comprehensive analyses. As NGS approaches typically yield millions of putatively representative reads per sample, efficient data management and visualization resources have become mandatory. Most usually, those resources are implemented through a dedicated Laboratory Information Management System (LIMS), solely to provide perspective regarding the available information. We developed an easily deployable web-interface, facilitating management and bioinformatics analysis of metagenomics data-samples. It was engineered to run associated and dedicated Galaxy workflows for the detection and eventually classification of pathogens. The web application allows easy interaction with existing Galaxy metagenomic workflows, facilitates the organization, exploration and aggregation of the most relevant sample-specific sequences among millions of genomic sequences, allowing them to determine their relative abundance, and associate them to the most closely related organism or pathogen. The user-friendly Django-Based interface, associates the users’ input data and its metadata through a bio-IT provided set of resources (a Galaxy instance, and both sufficient storage and grid computing power). Galaxy is used to handle and analyze the user’s input data from loading, indexing, mapping, assembly and DB-searches. Interaction between our application and Galaxy is ensured by the BioBlend library, which gives API-based access to Galaxy’s main features. Metadata about samples, runs, as well as the workflow results are stored in the LIMS. For metagenomic classification and exploration purposes, we show, as a proof of concept, that integration of intuitive exploratory tools, like Krona for representation of taxonomic classification, can be achieved very easily. In the trend of Galaxy, the interface enables the sharing of scientific results to fellow team members.
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Correia, Damien, Olivia Doppelt-Azeroual, Jean-Baptiste Denis, Mathias Vandenbogaert e Valérie Caro. "MetaGenSense: A web-application for analysis and exploration of high throughput sequencing metagenomic data". F1000Research 4 (1 dicembre 2016): 86. http://dx.doi.org/10.12688/f1000research.6139.3.
Testo completoAbstract (sommario):
The detection and characterization of emerging infectious agents has been a continuing public health concern. High Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies have proven to be promising approaches for efficient and unbiased detection of pathogens in complex biological samples, providing access to comprehensive analyses. As NGS approaches typically yield millions of putatively representative reads per sample, efficient data management and visualization resources have become mandatory. Most usually, those resources are implemented through a dedicated Laboratory Information Management System (LIMS), solely to provide perspective regarding the available information. We developed an easily deployable web-interface, facilitating management and bioinformatics analysis of metagenomics data-samples. It was engineered to run associated and dedicated Galaxy workflows for the detection and eventually classification of pathogens. The web application allows easy interaction with existing Galaxy metagenomic workflows, facilitates the organization, exploration and aggregation of the most relevant sample-specific sequences among millions of genomic sequences, allowing them to determine their relative abundance, and associate them to the most closely related organism or pathogen. The user-friendly Django-Based interface, associates the users’ input data and its metadata through a bio-IT provided set of resources (a Galaxy instance, and both sufficient storage and grid computing power). Galaxy is used to handle and analyze the user’s input data from loading, indexing, mapping, assembly and DB-searches. Interaction between our application and Galaxy is ensured by the BioBlend library, which gives API-based access to Galaxy’s main features. Metadata about samples, runs, as well as the workflow results are stored in the LIMS. For metagenomic classification and exploration purposes, we show, as a proof of concept, that integration of intuitive exploratory tools, like Krona for representation of taxonomic classification, can be achieved very easily. In the trend of Galaxy, the interface enables the sharing of scientific results to fellow team members.
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Ma, Zeqiang, Jason Gottwals, Hao Ho, Kristina J. Fasig, Heather Rietz, Taylor Hartley, Vladimir Kravstov et al. "Clinical Utility of High-Throughput and Complimentary Genomic Tumor Profiling in Hematologic Malignancies". Blood 126, n. 23 (3 dicembre 2015): 1388. http://dx.doi.org/10.1182/blood.v126.23.1388.1388.
Testo completoAbstract (sommario):
Abstract Background: NCCN guidelines and a continuously expanding collection of high-impact publications recommend interrogating hematologic neoplasms for biomarkers that yield diagnostic, prognostic, and/or therapeutic information. The advent of comprehensive, high-throughput genomic profiling technologies has enabled the detection of multiple genomic alterations in an efficient and cost effective manner, and provides insights into disease initiation, progression, therapy response and identification of therapeutic targets not available through conventional methods. Design: Based on NCCN guidelines and literature review, we designed a genomic profiling strategy composed of a targeted amplicon-based next generation sequencing (NGS) assay and cytogenomic microarray (CGA) to identify clinically actionable genomic alterations. In the current context, "actionable" was defined as helping establish a diagnosis through demonstration of "clonal hematopoiesis" as recommended by NCCN guidelines, informing prognosis, and/or providing a potential therapeutic target. These assays complement the routine work-up of hematologic tumors, which include flow cytometry, morphologic evaluation and FISH/cytogenetic analyses. Initial data from implementation of this testing strategy in our broad community-based practice are presented. Results: 865 patient samples were analyzed, which included the following: AML (168), ALL (24), MDS (194), MDS/MPN (34), CLL (82), MPN (156), samples with suspicion of a myeloid stem cell disorder (107) and others. Of the cases evaluated by NGS and CGA, genomic aberrations were detected in 70% and 48%, respectively. Conventional cytogenetic analyses revealed abnormalities in 38% of the cases for which conclusive results were obtained; abnormal FISH results were observed in 44.7%. In cases where conventional cytogenetics and FISH tests were negative, 70% were abnormal by either NGS or CGA (~81% in cases with evidence of a myeloid stem cell disorder or acute leukemia) (Figure 1). Importantly, 12% of 51 cases with normal FISH, cytogenetics and NGS results were abnormal by CGA, and 60% of 111 cases with normal FISH, cytogenetics and CGA results had actionable mutations detected by NGS. CGA and NGS aberrations were frequently detected in MPN or MDS/MPN cases with negative cytogenetics and FISH results. For example, among 156 MPN cases, CGA and NGS abnormalities were observed in 42% and 65% of cases respectively, while only 11% of cases had abnormal cytogenetics results and 10% of cases were FISH positive (Figure 2). In contrast, in cases without actionable mutations detected by NGS and CGA, which also had been analyzed by FISH/cytogenetics, 11% of 89 were cytogenetically abnormal, and 36% of 75 had genomic alterations detected by FISH. Conclusions: High throughput genomic tumor profiling through targeted DNA sequencing and analysis of copy number alterations complements conventional methods of tumor interrogation and leads to more frequent detection of actionable alterations. This is especially apparent in the context of morphologic and/or clinical suspicion of a myeloid stem cell disorder. These data indicate that integrating multiple strategies to identify informative biomarkers can enhance diagnosis, prognosis and/or therapy in hematologic disorders. Most importantly, we have observed changes in clinical management decisions across different disease states including AML, ALL, other myeloid malignancies, and CLL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ma: PathGroup: Employment. Gottwals:PathGroup: Employment. Ho:PathGroup: Employment. Fasig:PathGroup: Employment. Rietz:PathGroup: Employment. Hartley:PathGroup: Employment. Kravstov:PathGroup: Employment. Spence:PathGroup: Employment. Connor:PathGroup: Employment. Turnicky:PathGroup: Employment. Prescott:PathGroup: Employment. Lennon:PathGroup: Employment. Sathanoori:PathGroup: Employment. Flinn:Celgene Corporation: Research Funding. Berdeja:Celgene: Research Funding; Takeda: Research Funding; Curis: Research Funding; Abbvie: Research Funding; Onyx: Research Funding; MEI: Research Funding; Acetylon: Research Funding; Novartis: Research Funding; Array: Research Funding; Janssen: Research Funding; BMS: Research Funding. Wheeler:PathGroup: Employment. Coldren:PathGroup: Employment. Chandra:PathGroup: Employment. Mukherjee:PathGroup: Employment. Casey:PathGroup: Employment.
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Wesołowski, Wojciech, Beata Domnicz, Joanna Augustynowicz e Marek Szklarczyk. "VCF2CAPS–A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset". PLOS Computational Biology 17, n. 5 (20 maggio 2021): e1008980. http://dx.doi.org/10.1371/journal.pcbi.1008980.
Testo completoAbstract (sommario):
Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.
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Thomson, Emma, Camilla L. C. Ip, Anjna Badhan, Mette T. Christiansen, Walt Adamson, M. Azim Ansari, David Bibby et al. "Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes". Journal of Clinical Microbiology 54, n. 10 (6 luglio 2016): 2470–84. http://dx.doi.org/10.1128/jcm.00330-16.
Testo completoAbstract (sommario):
Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.
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Khan, Anwar, Nagehan Pakasticali, Omar Fathalla, Taiga Nishihori e Mohammad O. Hussaini. "Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma". Blood 138, Supplement 1 (5 novembre 2021): 1625. http://dx.doi.org/10.1182/blood-2021-154418.
Testo completoAbstract (sommario):
Abstract Introduction: Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices. Methods: We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review. 10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter). clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences Statistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis. Results: 192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1). Conclusion: Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. Figure 1 Figure 1. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.
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Harris, Marian, Donna S. Neuberg, Jianbiao Zheng, Malek Faham, Stephen E. Sallan e Lewis B. Silverman. "Minimal Residual Disease Detection Using High-Throughput Sequencing Predicts Clinical Outcome in Patients with Pediatric B-Lineage Acute Lymphoblastic Leukemia". Blood 124, n. 21 (6 dicembre 2014): 2391. http://dx.doi.org/10.1182/blood.v124.21.2391.2391.
Testo completoAbstract (sommario):
Abstract Background: Minimal residual disease (MRD) assessment at the end of induction is critical for risk stratification of B-lineage acute lymphoblastic leukemia (B-ALL) patients. There are multiple techniques for molecular MRD assessment, including next-generation sequencing (NGS) and allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin (Ig) or T-cell receptor rearrangements. Leukemia-specific rearrangements can be identified in >97% of B-ALL patients using the NGS-based LymphoSIGHT™ method, while ASO-PCR can typically identify leukemia-specific rearrangements in only 80-90% of B-ALL patients. In this report, we assessed the ability of the NGS method to predict event-free survival (EFS) in a cohort of B-ALL patients who were inevaluable by ASO-PCR. Methods: All patients were treated between 2005-2011 on a clinical trial conducted by the Dana-Farber Cancer Institute (DFCI) ALL Consortium (05-001) on which high levels of end-induction MRD (≥10-3) led to intensified treatment. The 37 pediatric B-ALL patients included in this study were inevaluable by ASO-PCR, and therefore this cohort represents a subset of patients in whom a decision to intensify therapy per protocol could not be made. Archived DNA samples from diagnosis and Day 32 were provided for MRD assessment using the NGS method. Using universal primer sets, we amplified the immunoglobulin (IGH-VDJ, IGH-DJ and IGK) and T cell receptor (TRB, TRD, TRG) loci in diagnostic bone marrow samples. Leukemia-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell and/or T-cell repertoire. The presence of leukemia-specific clonotypes was then assessed in the end-induction (Day 32) sample. NGS MRD assessment was performed blinded to all clinical and ASO-PCR information on these patients. The association of NGS MRD results and clinical outcome (EFS) was examined, and significance assessed using the log-rank test. Results: Of 37 patients who were inevaluable for end-induction MRD by ASO-PCR, leukemia-specific clonotypes were successfully identified in 35 (95%) by the NGS method. The use of a threshold of 10-3, which is the ASO-PCR threshold used for risk-stratification on DFCI protocols, demonstrated significantly improved EFS between the MRD negative and MRD positive groups (p<0.0001) (Figure 1A). A similar analysis with a threshold of 10-4 also demonstrated significantly improved EFS between the MRD negative and MRD positive groups (p<0.0001) (Figure 1B). Conclusions: This study demonstrates the prognostic significance of NGS-based MRD assessment in pediatric patients with B-ALL. This NGS-based assay led to successful assessment of MRD in 95% of patients who were inevaluable by ASO-PCR. The NGS method represents an alternative approach for clinical MRD monitoring that could fundamentally improve the ability to identify high-risk B-ALL patients and potentially improve their outcome. Figure 1A. Kaplan-Meier analysis of event-free survival for A) 30 MRD negative (<10-3) and 5 MRD positive (≥10-3) patients and B) 26 MRD negative (<10-4) and 9 MRD positive (≥10-4) patients. Figure 1A. Kaplan-Meier analysis of event-free survival for A) 30 MRD negative (<10-3) and 5 MRD positive (≥10-3) patients and B) 26 MRD negative (<10-4) and 9 MRD positive (≥10-4) patients. Figure 1B Figure 1B. Disclosures Zheng: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Tierno, Domenico, Gabriele Grassi, Serena Scomersi, Marina Bortul, Daniele Generali, Fabrizio Zanconati e Bruna Scaggiante. "Next-Generation Sequencing and Triple-Negative Breast Cancer: Insights and Applications". International Journal of Molecular Sciences 24, n. 11 (2 giugno 2023): 9688. http://dx.doi.org/10.3390/ijms24119688.
Testo completoAbstract (sommario):
The poor survival of triple-negative breast cancer (TNBC) is due to its aggressive behavior, large heterogeneity, and high risk of recurrence. A comprehensive molecular investigation of this type of breast cancer using high-throughput next-generation sequencing (NGS) methods may help to elucidate its potential progression and discover biomarkers related to patient survival. In this review, the NGS applications in TNBC research are described. Many NGS studies point to TP53 mutations, immunocheckpoint response genes, and aberrations in the PIK3CA and DNA repair pathways as recurrent pathogenic alterations in TNBC. Beyond their diagnostic and predictive/prognostic value, these findings suggest potential personalized treatments in PD -L1-positive TNBC or in TNBC with a homologous recombination deficit. Moreover, the comprehensive sequencing of large genomes with NGS has enabled the identification of novel markers with clinical value in TNBC, such as AURKA, MYC, and JARID2 mutations. In addition, NGS investigations to explore ethnicity-specific alterations have pointed to EZH2 overexpression, BRCA1 alterations, and a BRCA2-delaAAGA mutation as possible molecular signatures of African and African American TNBC. Finally, the development of long-read sequencing methods and their combination with optimized short-read techniques promise to improve the efficiency of NGS approaches for future massive clinical use.