Accurate Detection Of MtDNA Based On Next Generation Sequencing And Its Application As A Novel Biomarker Of Hepatocellular Carcinoma

Background: Hepatocellular carcinoma(HCC)is a malignant disease that seriously endangers the health of our people.At present,the biomarkers for diagnosis,prognosis and efficacy monitoring of HCC are far from enough.The traditional detection methods are mainly based on imaging,pathology and serological judgment.These methods are invasive and usually have low specificity and sensitivity.Moreover,real-time dynamic monitoring is difficult to achieve.The development of novel biomarkers to prolong the survival of patients is an urgent need for clinical practice.Due to the small genome,high copy number,high mutation rate and close relationship with the development of HCC,the precise detection of mitochondrial DNA(mt DNA)mutation and copy number variation has unique advantages as novel biomarkers.Next generation sequencing(NGS) technology has been widely used for mt DNA sequencing.However,it has been reported that there may be cross-contamination in mt DNA next-generation sequencing of multiple samples,which seriously interferes with the detection accuracy of mt DNA.Therefore,it is important to eliminate cross-contamination and establish accurate technologies of mt DNA detection.In addition,liquid biopsy technology represented by circulating tumor DNA(ct DNA)detection has developed rapidly,and it has provided sufficient technical support and experience for the development of cell-free mitochondrial DNA(cfmt DNA)related biomarkers.Purpose: 1.We plan to establish cross-contamination analysis and removal process in mt DNA next-generation sequencing of multiple samples and develop accurate detection technology of mt DNA;2.We plan to detect mt DNA mutation in HCC tissue and develop biomarkers for prognostic prediction;3.We plan to detect cell-free mt DNA copy number in HCC patients and develop biomarkers to diagnose HCC,predict prognosis and monitor Trans-arterial chemoembolization(TACE)efficacy.Methods: In the first part: Based on the dual-barcode library construction,I performed mt DNA sequencing of 240 samples.The Hem I 1.0-Heatmap illustrator was used to draw cross-contamination heat-maps,and then I systematically analyze the effects of different Illumina NGS platforms and capture methods on cross-contamination level in mt DNA sequencing.By comparing mt DNA mutations and haplogroup before and after filtering contamination,the interference of cross-contamination on mt DNA detection was analyzed.In the second part: Dual-barcode mt DNA sequencing technology was used to detect somatic mt DNA mutations in HCC and non-HCC tissues of 156 hepatitis B-related HCC(HBV-HCC)patients.Kaplan-Meier survival analysis of mt DNA mutations was performed in combination with prognostic information.Then,the critical contributing role of somatic D-loop mt DNA heteroplasmic mutations in HBV-related hepatocarcinogenesis was explored in vitro experiments.6 HCC cell lines with or without D-loop mutations were used to explore the biological functions and mechanisms;In the third part: Fragment size and content of plasma cell-free mt DNA(cfmt DNA)were analyzed with Picard.We evaluate the impact of library prepareation,coverage depth,and bioinformatics analysis on detection of cfmt DNA copy number.Then,feasibility of cfmt DNA copy number as a marker for HCC was explored.Results: In the first part: Based on the traditional single-barcode mt DNA sequencing strategy,there were different degrees of cross-contamination.On the Hiseq 2500 sequencing platform,the cross-contamination levels of 20 DNA samples ranged from 0.27% to 11.90%,and on Hiseq X Ten,from 0.93% to 17.70%.Our dual-barcode sequencing strategy could effectively eliminate these cross-contamination.In addition,our results showed that cross-contamination was mainly derived from capture and affected by different NGS platforms.The cross-contamination level on the Hiseq X Ten was significantly higher than that on the Hiseq 2500.Besides,cross-contamination level derived from capture and sequencing were both negatively correlated with sequencing coverage depth.Moreover,cross-contamination significantly increased false positives of mt DNA heteroplasmic mutations and interfered with heteroplasmic levels,but it had no significant effect on haplogroup classification of mtDNA.In the second part: There were a large number of mt DNA mutations in the HCC and non-HCC tissues of HBV-HCC patients.However,two mutation patterns were quite different.Although the average number and recurrent frequency of D-loop mutations in HCC tissues were lower than those in non-HCC tissues,the level of heteroplasmic was significantly higher.This suggested that mutations in D-loop region might be positively selected during the development of HCC.Multi-regional sequencing of 12 HCC patients further highlighted the positive selection of D-loop region mutations in HCC tissues.In addition,Kaplan-Meier survival analysis revealed that D-loop region mutations in HCC tissues were closely related to low mt DNA copy number and prognosis of HCC patients.In the vitro experiment,we initially explored the mechanism that D-loop mutations affected the prognosis of HBV-HCC patients.Our findings provide a possibility that D-loop region mutations in tumor tissues might influence proliferation,invasion and metastasis of HCC cell and thus the prognosis of HCC patients through reducing mt DNA copy number.These results revealed the critical contributing roles of D-loop mutations in HBV-related hepatocarcinogenesis.In the third part: In the cf DNA genome-wide sequencing data of hepatitis and HCC patients,the fragment size of the reads aligned to hg19 is mainly concentrated near 160 bp,and the fragment size of the reads aligned to mitochondrial genome is concentrated around 70 bp.This suggested that the degration degree of cfmt DNA is more severe than nuclear genome.Cfmt DNA copy number was significantly higher in patients with HCC than that in patients just with hepatitis(6.98 vs.2.64,P=0.002).The area under the curve(AUC)of cfmt DNA copy number in the HCC diagnosis was as high as 0.843,initially suggesting the potential of cfmt DNA as a diagnostic marker for HCC.According to the m RECIST criteria,the efficacy of TACE in 6 patients could be classified into 1 with partial remission(PR),4 with stable disease(SD),and 1 with disease progression(PD).The cfmt DNA copy number of patients with partial remission decreased from 8.38 to 6.30 after TACE,and the cfmt DNA copy number of patients with disease progression increased from 12.83 to 35.80 after TACE.Overall,changes in plasma cfmt DNA copy number after TACE were consistent with imaging findings.Conclusions : The novel dual-barcode sequencing strategy effectively eliminates cross-contamination in mt DNA sequencing and improves the accuracy of mt DNA detection.Based on capture-based NGS technology,whole mt DNA sequencing with ultra-deep coverage depth is performed in 156 HBV-related HCC patients to profile mt DNA somatic mutations.Our data elucidate the critical contributing role and underlying mechanism of heteroplasmic mt DNA D-loop mutations in HBV-related hepatocarcinogenesis,suggesting that mt DNA D-loop mutation have potential for predicting prognosis of HBV-HCC.Preliminary results demonstrate the feasibility of cfmt DNA as a HCC marker for the diagnosis,prognosis and efficacy monitoring.