Method for analyzing a biological material sample using a pipetting station to detect mosaic genetic variants with a very low frequency of occurrence in genes

Abstract

The subject of the application is a method of analyzing a sample of biological material using a pipetting station in order to detect mosaic genetic variants with a very low frequency of occurrence in genes, consisting in isolating DNA from a peripheral blood sample and then preparing DNA libraries for duplex sequencing and further bioinformatic processing, characterized in that a) DNA samples are prepared and mechanical DNA fragmentation is performed using an ultrasonicator; b) the obtained DNA fragments of about 500 base pairs in length are subjected to enzymatic reactions, adapter ligation, incubation and purification using magnetic beads, wherein the enzymatic reactions include repair of the ends of the fragmented DNA, attachment of dedicated adapters, amplification by means of PCR, and then; c) amplification and hybridization of dedicated molecular probes of a region of the human genome are performed;then d) the quality and quantity of the obtained DNA libraries are verified, after which they are sequenced, and the obtained results are analyzed using bioinformatics tools, obtaining at the final stage of analysis a list of genetic variants along with the frequency of their occurrence in the examined gene region, and the obtained results are analyzed using bioinformatics tools, obtaining at the final stage of analysis a list of genetic variants along with the frequency of their occurrence in the examined gene region, whereby stages b) and c) are carried out using the Bravo NGS station from Agilent Technologies;