Academic literature on the topic 'Sanger/pyrosequencing'
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Journal articles on the topic "Sanger/pyrosequencing"
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Tewari, Deepanker, Stephen Cieply, and Julia Livengood. "Identification of bacteria recovered from animals using the 16S ribosomal RNA gene with pyrosequencing and Sanger sequencing." Journal of Veterinary Diagnostic Investigation 23, no. 6 (2011): 1104–8. http://dx.doi.org/10.1177/1040638711425583.
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Bacterial identification using genetic sequencing is fast becoming a confirmatory tool for microbiologists. Its application in veterinary diagnostic laboratories is still growing. In addition to availability of Sanger sequencing, pyrosequencing has recently emerged as a unique method for short-read DNA sequencing for bacterial identifications. Its ease of use makes it possible to diagnose infections rapidly at a low cost even in smaller laboratories. In the current study, pyrosequencing was compared with Sanger sequencing for identification of the bacterial organisms. Fifty-four bacterial isolates spanning 23 different bacterial families encountered in veterinary diagnostic microbiology laboratories were sequenced using 16S ribosomal RNA gene with pyrosequencing and Sanger sequencing. Pyrosequencing was able to identify 80% of isolates to the genus level, and 43% isolates to the species level. Sanger sequencing with approximately 500 bp performed better for both genus (100%) and species (87%) identification. Use of different sequence databases to identify bacteria isolated from animals showed relative importance of public databases compared to a validated commercial library. A time and limited cost comparison between pyrosequencing and genetic sequencing of 500 bp showed pyrosequencing was not only faster but also comparable in cost, making it a viable alternative for use in classifying bacteria isolated from animals.
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Novak-Frazer, Lilyann, Samuel P. Anees-Hill, Darin Hassan, et al. "Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens." Journal of Antimicrobial Chemotherapy 75, no. 12 (2020): 3501–9. http://dx.doi.org/10.1093/jac/dkaa357.
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Abstract Background Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. Objectives To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. Methods Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. Results The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. Conclusions The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.
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Fakruddin, Md, and Abhijit Chowdhury. "Pyrosequencing-An Alternative to Traditional Sanger Sequencing." American Journal of Biochemistry and Biotechnology 8, no. 1 (2012): 14–20. http://dx.doi.org/10.3844/ajbbsp.2012.14.20.
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Schildgen, V., J. Lusebrink, C. Schulz, et al. "EGFR mutations: Comparison of Sanger- and pyrosequencing." Journal of Clinical Oncology 29, no. 15_suppl (2011): e21019-e21019. http://dx.doi.org/10.1200/jco.2011.29.15_suppl.e21019.
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Prigigallo, Maria I., Ahmed Abdelfattah, Santa O. Cacciola, et al. "Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing." Phytopathology® 106, no. 3 (2016): 305–13. http://dx.doi.org/10.1094/phyto-07-15-0167-r.
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A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.
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Wiederhold, Nathan P., Jodi L. Grabinski, Guillermo Garcia-Effron, David S. Perlin, and Samuel A. Lee. "Pyrosequencing To Detect Mutations in FKS1 That Confer Reduced Echinocandin Susceptibility in Candida albicans." Antimicrobial Agents and Chemotherapy 52, no. 11 (2008): 4145–48. http://dx.doi.org/10.1128/aac.00959-08.
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ABSTRACT Pyrosequencing was compared to Sanger dideoxy sequencing to detect mutations in FKS1 responsible for reduced echinocandin susceptibility in Candida albicans. These methods were in complete agreement for 10 of 12 clinical isolates with elevated echinocandin MICs, supporting the potential feasibility of pyrosequencing to detect mutations within diploid fungi.
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Lüke, Claudia, and Peter Frenzel. "Potential ofpmoAAmplicon Pyrosequencing for Methanotroph Diversity Studies." Applied and Environmental Microbiology 77, no. 17 (2011): 6305–9. http://dx.doi.org/10.1128/aem.05355-11.
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ABSTRACTWe analyzed the potential ofpmoAamplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogeneticpmoAlineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.
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Poulet, Axel, Maud Privat, Flora Ponelle, et al. "Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software." BioMed Research International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/5623089.
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Screening forBRCAmutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of theBRCAgenes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.
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Harrington, Colleen T., Elaine I. Lin, Matthew T. Olson, and James R. Eshleman. "Fundamentals of Pyrosequencing." Archives of Pathology & Laboratory Medicine 137, no. 9 (2013): 1296–303. http://dx.doi.org/10.5858/arpa.2012-0463-ra.
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Context.—DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. Objective.—To demonstrate the fundamental principles of pyrosequencing. Data Sources.—Salient features of pyrosequencing are demonstrated using the free software program Pyromaker (http://pyromaker.pathology.jhmi.edu), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results. Conclusions.—We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.
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Cardus, Beatrix, Richard Colling, Angela Hamblin, and Elizabeth Soilleux. "Comparison of methodologies for the detection of BRAF mutations in bone marrow trephine specimens." Journal of Clinical Pathology 72, no. 6 (2019): 406–11. http://dx.doi.org/10.1136/jclinpath-2019-205734.
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AimsBRAF V600E detection assists in the diagnosis of hairy cell leukaemia (HCL); however, testing practices vary. We evaluated the clinical utility of 5 BRAF mutation testing strategies for use on bone marrow trephines (BMT).Methods11 HCL, 5 HCL ‘mimic’, 2 treated HCL and 10 normal BMT specimens were tested for mutant BRAF, comparing Sanger sequencing, pyrosequencing, amplicon-based next generation sequencing (NGS), automated (Idylla) PCR and immunohistochemistry (IHC).ResultsPCR and IHC were cheaper and identified V600E in 100 % of HCL cases. Pyrosequencing detected the mutation in 91%, NGS in 55% of cases and Sanger sequencing in 27%. All assays gave wild-type BRAF results in HCL mimics and normal BMT samples.ConclusionsPCR and IHC were most sensitive and cost-effective, but these have limited scope for multiplexing and are likely to be replaced by NGS gene panels or whole genome sequencing in the medium to long term.
Dissertations / Theses on the topic "Sanger/pyrosequencing"
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Alessandrini, Gaia. "Metodi innovativi per il sequenziamento di acidi nucleici." Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amslaurea.unibo.it/7341/.
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Martinsson, Caroline. "Characterisation of EGFR and KRAS mutations in non-small cell lung cancer." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-126041.
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Background: Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with EGFR mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with KRAS mutations do not benefit of such treatment. Methods: This study investigates the frequency of EGFR and KRAS mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes. Results: Five EGFR mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. KRAS mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations. Conclusions: This study confirms previous observations regarding the frequency of EGFR and KRAS mutations in non-small cell lung cancer. Mutations in EGFR and KRAS were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.
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Forget, Nathalie. "Free-Living and Symbiotic Bacterial Communities in Contrasting Hydrothermally Active Habitats." Thesis, 2013. http://hdl.handle.net/1828/4869.
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Prokaryotic microorganisms, which are at the base of deep-sea hydrothermal vent food webs, adapt rapidly to environmental fluctuations. This study aimed at comparing bacterial communities in contrasting hydrothermal habitats to better understand compositional adaptations to local conditions. I first used small subunit (SSU) ribosomal RNA (rRNA) gene sequences to compare mat-forming bacterial communities associated with iron oxides at two hydrothermal vent sites on the Tonga Arc, southwest Pacific. Operational taxonomic units (OTUs), defined at 97% sequence similarity, were affiliated to a great diversity of autotrophic and heterotrophic groups. Metabolically diverse Gammaproteobacteria dominated the sample from Volcano 19, collected at 992 m depth. The sample from Volcano 1, collected at 197 m depth, was dominated by iron-oxidizing bacteria from the class Zetaproteobacteria. The depth of the sampling sites was proposed to explain clone library dissimilarities.
In the following studies, I compared bacterial communities associated with the vestimentiferan tubeworm Ridgeia piscesae, a foundation species at the Juan de Fuca Ridge. Samples of the polychaete were collected from tubeworm habitats in contrasting flow regimes that influenced temperature and hydrogen sulphide concentrations. Free-living bacteria were analyzed using both sequencing and 454 pyrosequencing of the SSU rRNA gene. Statistical analyses suggested a predictable pattern of bacterial community composition for the two habitats, with higher proportions of sulphur and hydrogen oxidizers in High Flow and more heterotrophic groups in Low Flow environments. Temperature, available energy for metabolism, and stability of the habitat were suggested to explain these distinctive bacterial communities. Symbiotic assemblages were investigated using the same sequencing methods together with catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Gammaproteobacteria dominated all sequence libraries, followed by Epsilonproteobacteria. CARD-FISH confirmed the co-occurrence of these groups within R. piscesae trophosomes. Statistical analyses indicated distinctive membership and structure of trophosome assemblages between sampling sites. Analysis of R. piscesae juvenile showed distinctive structural properties when compared to adult individuals, but similar membership, within sampling sites. These results suggested that the composition of trophosome assemblages might be affected by specific physical and chemical conditions at each vent site and that a selection process might occur during R. piscesae’s development.<br>Graduate<br>0410<br>0416<br>0329<br>nathalieforget@gmail.com
Book chapters on the topic "Sanger/pyrosequencing"
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Presneau, Nadège, and Rifat Hamoudi. "Sequencing Technologies." In Molecular Diagnostics. Oxford University Press, 2019. http://dx.doi.org/10.1093/hesc/9780198777069.003.0005.
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This chapter explores the technologies used for sequencing. It discusses the principles of first-generation sequencing, namely Sanger sequencing and pyrosequencing methods. The chapter also notes the principles of sequencing by synthesis (SBS) alongside the technologies developed for second-generation sequencing, that is, Illumina and Ion Torrent. Additionally, it discusses third-generation sequencing, which allows the real-time sequencing of single molecules, and the fourth-generation sequencing, which merged in situ cellular localization and gene expression sequencing. The chapter then highlights the main advantages and challenges associated with each sequencing technology and considers some of their applications.
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Mathews, Amit. "DNA Sequencing: A Brief History." In DNA Sequencing - History, Present and Future [Working Title]. IntechOpen, 2024. https://doi.org/10.5772/intechopen.1007844.
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DNA sequencing, which deciphers DNA nucleotide sequence, has had a transformative impact on biology and medicine. The quest began in 1953 with Watson and Crick’s discovery of the DNA double helix, based on Franklin and Wilkins’ pioneering work in X-ray crystallography. Arthur Kornberg’s 1956 discovery of DNA polymerase further advanced the field, laying the groundwork for future sequencing technologies. In 1977, Frederick Sanger’s chain-termination method (Sanger sequencing) and Maxam-Gilbert’s chemical sequencing emerged as the first viable techniques for reading DNA. Sanger sequencing, in particular, remains widely used for analyzing shorter DNA sequences today. The 1980s saw the introduction of automated sequencers, which dramatically boosted throughput, precision, and accessibility. Launched in 1990, The Human Genome Project (HGP) utilized Sanger sequencing to map the human genome, marking a historic achievement that is often compared to landing on the moon. The early 2000s ushered in next-generation sequencing (NGS) technologies, such as pyrosequencing, sequencing by synthesis (SBS), and SOLiD, offering faster and more cost-effective sequencing. Today, third-generation technologies like Oxford Nanopore’s “nanopore” sequencing and PacBio’s Single-Molecule Real-Time (SMRT) sequencing enable longer reads and real-time data. These advancements have revolutionized genomics, driving progress in evolutionary biology, precision medicine, and forensic science.
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Archibald, John. "2. How to read the book of life." In Genomics: A Very Short Introduction. Oxford University Press, 2018. http://dx.doi.org/10.1093/actrade/9780198786207.003.0002.
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For all its biological importance, DNA is a fragile molecule so extracting it is a difficult process. ‘How to read the book of life’ explains the techniques required to sequence DNA. It begins by explaining the techniques developed for protein and RNA sequencing by Frederick Sanger, Robert Holley, and Carl Woese that were then developed further for DNA sequencing. Following the success of the Human Genome Project, the next generation of DNA sequencing was developed in the mid-2000s. Pyrosequencing was capable of generating orders of magnitude more data at a fraction of the cost, but was superceded within a decade by semiconductor sequencing, reversible chain-termination sequencing, and single-molecule sequencing.
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Singh, Yengkhom Daniel, Thingujam Deeparani, and Babina Sarangthem. "CURRENT PRACTICE OF PATHOLOGY IN THE MOLECULAR ERA." In Futuristic Trends in Medical Science Volume 2 Book 22. Iterative International Publishers, Selfypage Developers Pvt Ltd, 2023. http://dx.doi.org/10.58532/v2bs22p1ch8.
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Molecular Pathology is defined by the Association of Clinical Pathologists as ―the study of molecules in a disease state‖ by using the tools of molecular biology to better understand the aetiology, pathogenesis, diagnosis and prognosis of diseases. The use of antibody integrated specific immunohistochemical stains marks the dawn of the Molecular era of pathology. This chapter discusses briefly the various modern biology tools used in diagnostics and the role of molecular pathologist in understanding diseases and malignancies in particular as a part of the multidisciplinary team consisting of pathologist, radiologist, clinico-oncologist, surgeon and therapist. The commonly used modern biology tools in diagnostics are Immunohistochemistry, Pyrosequencing, Sanger sequencing, Reverse transcriptase (RT)- PCR, q RT-PCR, FISH, Next-generation sequencing (NGS) targeted panels etc. Newer tools like Liquid biopsy, digital pathology, tissue biobanking, patients derived organoids are also briefly mentioned. The importance of integration of morphological pathology and molecular diagnostics and the need for trained Molecular Pathologist is emphasized here. Molecular pathology is currently of great importance, and is becoming increasingly significant as it enables more precise diagnosis and treatment selection. Hence, the ―molecular revolution‖ has deeply transformed cancer care, re-evaluating the role of the pathologic diagnoses as the backbone of the therapeutic decision-making process. Establishment of a multidisciplinary cancer institute with a single laboratory equipped with both diagnostics and research facilities is the need of the hour