| Abstract [eng] |
Next Generation Sequencing (NGS) is a nucleic acid sequencing process broadly used in medicine and scientific researches. One of the NGS application is a whole genome sequencing (WGS) method, in which the complete DNA sequence of the organism is determined. This method consists of three main steps: DNA library preparation, sequencing, and data analysis. The most important steps in the DNA library preparation phase are the DNA fragmentation and introduction of sequencing platform-specific DNA adaptors. While performing WGS it is often encountered with uneven genome coverage which depends on GC content of DNA (GC bias). According to the literature, one of the major causes for this occurrence is that DNA libraries are prepared using reactions, which require high temperature (~65 °C). When platform-specific adaptors are introduced during ligation process, DNA fragment ends first need to undergo dA-addition reaction that is performed at high temperature. Also if DNA fragmentation is performed with endonuclease, high temperature is required to inactivate the enzyme. This thesis focuses on strategy of endonuclease immobilization on solid surface to easily remove endonuclease from further reactions, thus avoiding thermal inactivation of enzyme or labour and time consuming purification of DNA from fragmentation mixture. The thesis also evaluates the possibility of performing the dA-addition reaction at a lower temperatures. |
