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Contribution of Next Generation Sequencing for Laboratory Diagnostics
Votýpka, Pavel ; Beránek, Martin (advisor) ; Macháček, Miloslav (referee)
5 ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavel Votýpka Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Consultant: Mgr. Nikola Ptáková Title of diploma thesis: Contribution of Next Generation Sequencing for Laboratory Diagnostics The endeavor to sequence the whole human genome lead not only to the knowledge acquisition regarding the human genetic information but as well to the development of new sequencing methods and technologies. In order to keep up with progress in genetic field in many clinical and research laboratories the new massive parallel sequencing equipment is being utilized. On the market are currently established four leading platforms - Illumina, Solid, Ion Torrent and 454 Life Technologies. The process of sequencing analysis can be summarized into three main steps - the sequencing library preparation, sequencing itself, variant calling and data analysis. Each part of the sequencing analysis exhibits certain specifics, we need to count with and as well its pitfalls, we need to avoid or to minimalize their impact on the analysis final result. Recently new methods termed sequencing of the 3rd generation are being developed, enabling sequence of a single DNA molecule to be determined without previous...
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Contribution of Next Generation Sequencing for Laboratory Diagnostics
Votýpka, Pavel ; Beránek, Martin (advisor) ; Macháček, Miloslav (referee)
5 ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavel Votýpka Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Consultant: Mgr. Nikola Ptáková Title of diploma thesis: Contribution of Next Generation Sequencing for Laboratory Diagnostics The endeavor to sequence the whole human genome lead not only to the knowledge acquisition regarding the human genetic information but as well to the development of new sequencing methods and technologies. In order to keep up with progress in genetic field in many clinical and research laboratories the new massive parallel sequencing equipment is being utilized. On the market are currently established four leading platforms - Illumina, Solid, Ion Torrent and 454 Life Technologies. The process of sequencing analysis can be summarized into three main steps - the sequencing library preparation, sequencing itself, variant calling and data analysis. Each part of the sequencing analysis exhibits certain specifics, we need to count with and as well its pitfalls, we need to avoid or to minimalize their impact on the analysis final result. Recently new methods termed sequencing of the 3rd generation are being developed, enabling sequence of a single DNA molecule to be determined without previous...
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Molecular genetic analysis of the CFTR gene in neonatal screening in the Czech Republic
Votýpka, Pavel ; Klusoňová, Hana (advisor) ; Libik, Malgorzata Dorota (referee)
6 Summary Cystic fibrosis (CF) is the most frequent serious autosomal recessive genetic disease. CF is caused by mutations in the gene coding for Chloride transporter on the cell membrane (66). According to the statistics, this disease has prevalence 1 in 2500 to 3500 live newborns in European populations, where in Czech republic about 40 affected children a year are born (63). With respect to very serious clinical symptoms of this disorder (which substantially shortens the life expectancy of affected individual) in all newborns born in Czech Republic since October 1, 2009 the newborn screening from the dry blot spot is being performed. Blood samples from newborns who have in the primary test increased levels of IRT over given cut off are being sent to molecular genetic laboratory for analysis of the most frequent, pathogenic and population-wise important mutations in the CFTR gene. DNA isolation is being performed from the same dry blood spots on the screening card, which correspond to those where the increased IRT were found. It is important to choose the most suitable isolation method with respect to DNA yield and concentration. Those are crucial for quality and turn around time of molecular genetic analysis results and influence the following information retrieval . There are 2 mutations found in...
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