Study Of Cell Free Mitochondrial DNA Mutations By Targeted Capture-Based Sequencing

BACKGROUND In recent years,liquid biopsy technology has made a significant breakthrough in the clinical detection and application of new tumor markers.To achieve the purpose of diagnosis or monitoring of tumors.it mainly detects circulating tumor cells(CTCs),circulating tumor DNA(ct DNA),or exosomes released by the primary tumor or metastases in peripheral blood or other body fluids through various technical methods.Mitochondria is important organelles found in almost all eukaryotic cells.Mitochondria DNA(mt DNA)is a double-stranded closed loop structure that includes coding and non-coding regions.The total length is only 16569 bp.The amount of data generated by sequencing is small,so the detection is fast and low cost.mt DNA has a high copy number,and a cell can contain hundreds to thousands of copies of mt DNA.In addition,mt DNA in tumor cells often exists in the environment of high levels of reactive oxygen species,lacks histone protection and an effective DNA repair system,so its frequency of mutation is about 10 times higher than nuclear genome.Therefore,peripheral blood cell free mt DNA has clinical application value as a diagnostic and therapeutic marker,and is expected to become a new tumor biomarker for liquid biopsy.OBJECTIVES 1.To analyze the characteristics of cell-free mitochondrial DNA;2.To establish a large-scale accurate detection method for cell-free mt DNA mutations in tumor patients;3.To observe the characteristics of cell-free mt DNA mutations in tumor patients,and explore mt DNA mutations that may play important roles in tumorigenesis and evolution.METHODS 1.Construct a whole-genome sequencing library,analyze the copy number,fragment distribution characteristics,and breakpoint characteristics of cell-free mt DNA in plasma by sequencing;2.Capture mt DNA from a whole-genome sequencing library with a homemade mitochondrial DNA hybridization capture probe,and observe the differences effects of probe usage,the length of probe,hybridization temperature,and number of amplifications on capture efficiency to optimize capture experiment procedures;3.Use the optimized procedures described above to capture cell-free mt DNA in tumor patients,and use self-built bioassays to analy somatic mutations in cell-free mt DNA and profile the mt DNA mutation.RESULTS 1.The copy number of cell-free mt DNA is significantly lower than that of tissue mt DNA;the fragment distribution is concentrated between100-140 bp,which is significantly lower than that of cell-free mt DNA,and the breakpoints are regularly distributed;The capture efficiency increases first and then decreases with the increase in probe usage.Probe length,hybridization temperature,and number of amplifications all affected the capture efficiency.Optimized procedures have been established by setting different experimental parameters;3.The number of mt DNA heterogeneous mutations in the plasma is higher than tumor tissues;4.The plasma of tumor patients has the same mt DNA heterogeneous mutations as the tumor tissue.CONCLUSION There is a difference of mt DNA mutations between plasma and tumor tissues,and experimental parameters should be personalized to improve capture efficiency.Based on targeted capture sequencing technology,tumor-derived mt DNA mutations can be accurately detected in plasma.