Document Details

Document Type : Thesis 
Document Title :
Establishment of Next-Generation Sequencing Assay as a Diagnostic Tool for Infectious Disease
استحداث فحص لتشخيص الأمراض المعدية باستخدام تقنية الجيل التالي لتسلسل الحمض النووي
 
Subject : Faculty of Science 
Document Language : Arabic 
Abstract : Background: Globally, infectious diseases are still the leading cause of human morbidity and mortality. The main limitation to prevent and minimize the burden of infectious disease is identifying the etiological agent associated with the infection. Clinical metagenomics (CMg) is a promising approach that helps to identify etiological agents in cases of unknown infections. The application of CMg in clinical settings still requires an optimization of the sample preparation steps to be used in Next-Generation Sequencing platforms prior to introducing it into clinical laboratories. The study aimed to establish‎ an assay for CMg targeting different taxonomy of etiological agents, whether virus, bacteria or fungus, in the clinical sample contrary to what is available in this regard. Methods: A mock sample spiked with five different pathogens, which were C. neoformans, K. pneumoniae‎‎‎, S. aureus‎‎, ALKV and AdV, was used for the comparative evaluation of different protocols belong to nucleic acid extraction step and human DNA depletion step. Also, the same mock sample was used to test the choices of digestion or not the DNA prior reverse transcription (RT) step and the primers used in this step. All the resultants were subjected to library preparation and loaded on the MiSeq platform. The analysis was done using GENEIOUS software to calculate the numbers of read belong to each spiked pathogen and how much of these reads covering from its reference genome. Finally, the assay was assembled and applied on clinical samples with single or multiple known infections. Results: the PowerViral® Environmental RNA-DNA Isolation Kit with a cooled 5-min bead beating and NEBNext Microbiome DNA Enrichment Kit were selected based on the outcome of their comparative evaluation. Also, ignoring DNA digestion step and using the non-ribosomal primers in the RT step to deplete rRNA were chosen for the metagenomic assay. Conclusion: The applying of the established metagenomic assay in this study on clinical specimens especially the multi-infectious ones showed the ability to successfully identify different types of pathogens. Recommendation: The established metagenomic assay in this study should be applied on a large scale of clinical samples with known infections prior using it with unknown infections. 
Supervisor : Prof. Taha A. Kumosani 
Thesis Type : Doctorate Thesis 
Publishing Year : 1442 AH
2020 AD 
Co-Supervisor : Prof. Jehad M. Yousef 
Added Date : Saturday, February 27, 2021 

Researchers

Researcher Name (Arabic)Researcher Name (English)Researcher TypeDr GradeEmail
سها عبد العال فراجFarraj, Suha AbdulaalResearcherDoctorate 

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