@phdthesis{Schmidt2022, author = {Schmidt, Jonas Paul Christian}, title = {Nanopore sequencing in laboratory medicine : novel molecular diagnostic prospects for Familial Mediterranean fever and SARS-CoV-2 infections}, doi = {10.26127/BTUOpen-6384}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:co1-opus4-63847}, school = {BTU Cottbus - Senftenberg}, year = {2022}, abstract = {Nanopore sequencing, a third-generation sequencing technique that applies nanometre sized pores to transduce the physical and chemical properties of specific nucleobases into measurable electrical signals, shows attractive advantages over conventional next-generation sequencing techniques. However, primarily due to high sequencing error rates this technique has rarely been used so far in clinical laboratory diagnostics. In this cumulative dissertation Nanopore sequencing was established and validated in clinical diagnostics using the example of the molecular diagnosis of Familial Mediterranean fever (FMF) and SARS coronavirus-2 (SARS-CoV-2) infections. First, a novel data analysis pipeline for accurate single nucleotide polymorphism (SNP) genotyping using Nanopore sequencing data was developed and validated with the corresponding sequencing protocol against conventional Sanger sequencing using 47 samples of patients with clinical suspicion of FMF. This method comparison showed a perfect agreement between both methods rendering current Nanopore sequencing in principle suitable for SNP genotyping in human genetics. The bioinformatic analysis of sequencing data is one of the most challenging parts in Nanopore sequencing experiments and complicates the application in a clinical diagnostic setting. Therefore, six different bioinformatic tools for sequence alignment were evaluated regarding their applicability to Nanopore sequencing data. This evaluation revealed a good suitability of all except one of these tools although differences in quality and performance exist. Since Nanopore sequencing showed a robust performance in SNP genotyping, a SARS-CoV-2 whole genome sequencing (WGS) protocol was established to enable onside viral WGS in a clinical laboratory. This was especially important for viral molecular biological surveillance during the pandemic as shown by analysing viral genetic data over the course of one year. Applying this approach in a clinical research project to investigate host-virus interaction by aggregating for the first time viral genetic data, serological data and clinical data, showed diverse humoral immune responses to SARS-CoV-2, that appear to be influenced by age, obesity and disease severity. Further, even small viral genetic changes may influence the clinical presentation of the associated disease COVID-19. Additionally, a novel reverse transcriptase (RT)- loop mediated isothermal amplification (LAMP) assay for the detection of SARS-CoV-2 was developed and validated for diagnostic use by method comparison with conventional RT-polymerase chain reaction (PCR). In summary, by presenting advancements of sequencing and bioinformatic workflows with the focus on an application in clinical diagnostics, the results of this thesis may pave the way for a broader application of Nanopore sequencing in laboratory medicine in the near future.}, subject = {Nanopore sequencing; Laboratory medicine; Molecular diagnostics; Familial Mediterranean fever; SARS-CoV-2; Nanopore-Sequenzierung; Labormedizin; Molekulare Diagnostik; Famili{\"a}res Mittelmeerfieber; Famili{\"a}res Mittelmeerfieber; Labormedizin; Molekulare Diagnostik; Sequenzierung; SARS-CoV-2; Diagnostics; Molecular medicine}, language = {en} }