Comparative Analysis Of Whole Exome Sequencing Of Cardia Adenocarcinoma,adjacent Cancer And Peripheral Blood In The Same Individual

1.Backgroud and objectiveThe amount of data of exons in human genome only accounts for 1%(about 30 MB)of the total.We can detect more low-frequency mutations and rare mutations than the whole genome.At the same time,we can greatly reduce the cost of sequencing and shorten the experimental period.Wes is the most effective method to find,identify and screen tumor related pathogenic genes.Through this technology,many pathogenic genes of human diseases have been found,and some of them have become the target genes for early diagnosis,prevention and treatment.At present,WES has been used in different fields of tumor research,but there is always a dispute about how to choose normal control in tumor field.The current tumor WES studies usually choose one of the peripheral blood DNA or the DNA of the adjacent tissues as the control,but often have inconsistent results.In addition,for the gastrointestinal cavity like organ tumors,although the adjacent tissue morphology is normal,many studies have found that the normal epithelial tissue actually has molecular changes.Through literature research,we found that the comparative analysis of Wes in the same individual cardiac adenocarcinoma,paracancerous and peripheral blood has not been reported.The purpose of this paper is to provide guidance for the selection of normal control group of cardiac adenocarcinoma.2.Materials and methods2.1 Sample preparationThe study obtained the informed consent of patients and their families and the review and approval of relevant institutions.We collected 10 pairs of cardiac adenocarcinoma tissue,paracancerous tissue and peripheral blood from the same individual.The samples were stored in-80? refrigerator.2.2 Experimental flow of whole genome exon sequencingDetection and quality control of DNA samples?breaking DNA into small fragments with a crusher?repairing,phosphorylating and adding a tail at the end of DNA?connecting the joints?capturing the sequence of exon region with probe hybridization?getting out the unbound DNA fragments?quality detection of DNA library?sequencing on the machine of hiseq pe1502.3 Analysis method2.3.1 Quality evaluation of sequencing dataThrough the statistical evaluation of the error rate,contrast rate and data volume of the original sequencing data,the subsequent analysis can only be carried out after meeting the standards,otherwise,the database and additional test need to be built again.2.3.2 Collection and analysis of variation informationThe original sequencing sequence of high quality was compared to the reference gene sequence of human,the variation information of germline level in the samples was detected,the somatic mutation was detected in the paired samples of cardiac adenocarcinoma,and the somatic mutation and high-frequency mutation gene were analyzed.3.Results3.1 Number of somatic mutations in cardia adenocarcinoma vs.paracancerous,cardia adenocarcinoma vs.peripheral blood,and paracancerous vs.peripheral bloodGastric cardia adenocarcinoma tissue vs.side-to-cancer tissue(T vs.N)sequencing of the entire genome.Gastric cardia adenocarcinoma tissues found 8525 SNV,131 InDel,and 1150 CNV.Gastric cardia adenocarcinoma tissue vs.peripheral blood(T vs.B)for whole-genome exons Sequencing revealed 8674 SNVs,134 InDels,and 625 CNVs in gastric cardia adenocarcinoma tissues;whole-genome exon sequencing was performed in paracancerous vs peripheral blood(N vs.B),and 4297 SNVs,42 InDel,and 362 were found in paracancerous tissue CNV.3.2 Number of SNV genes in the same individual with cardia adenocarcinoma vs.pericarcinoma,cardia adenocarcinoma vs.peripheral blood,and paracarcinoma vs.peripheral blood(mutation frequency VAF>0.05)In gastric cardia adenocarcinoma tissue vs.paracancerous tissue(T vs.N),there were 2390 genes with SNV mutation in the gastric cardia adenocarcinoma tissue and VAF greater than 0.05;in the cardia adenocarcinoma vs peripheral blood(T vs.B),SNV mutation occurred in the cardia adenocarcinoma tissue and The number of genes with VAF greater than 0.05 was 2433;in the peripheral blood(N vs.B),SNV mutations occurred in paracancerous tissue and the number of genes with VAF greater than 0.05 was 785.3.3 The same SNV gene in gastric cardia adenocarcinoma tissue vs.peripheral blood and paracancerous tissue vs.peripheral bloodCompared with the peripheral blood of cardiac adenocarcinoma vs.peripheral blood and paracancer tissue vs.peripheral blood of the same individual,the same SNV gene occurred in T and N of samples G070,g072,g074 and g075.The common SNV gene in T and N of samples G070 was SFN,znf746,TDG and tp53tg3d;the common SNP gene in T and N of samples g072 was fahd2b;the common SNP gene in T and N of samples g074 was simc 1.FoxO3,Slc2a3,TDG,krtap3-3,cstfl;the common single nucleotide mutations of T and N in g075 samples are csdel,or2t4,or2t2,amigo3,HTR1B,PTEN,prh2,c17orf97,Rockl,tmc4,znf416,PABPC1.3.4 High frequency mutations in gastric cardiac adenocarcinoma tissue vs.peripheral bloodT vs.B found 19 high-frequency mutation genes:TP53(75%),ZNF208(38%),CNTN6(38%),RBM42(25%),YBX3(25%),UNC80(25%),TRIM64C(25%),RENL(25%),RBBP8NL(25%),CYP8B1(25%),CAAP1(25%),ANKRD30A(25%),SPAG16(12%),PANK2(12%),NAT6(12%),NARFL(12%),KIR3DL1(12%),DMRTB1(12%),CFHR2(12%).3.5 High frequency mutation gene of gastric cardiac adenocarcinoma tissue vs.paracancer tissueT vs.N found 15 high-frequency mutation genes:TP53(62%),ZNF208(38%),CNTN6(38%),YBX3(25%),TRIM64C(25%),RBBP8NL(25%),CYP8B1(25%),SPAG16(12%),RBM42(12%),PANK2(12%),NAT6(12%),NARFL(12%),KIR3DL1(12%),DMRTB1(12%),CFHR2(12%).3.6 High-frequency mutations in paracancer tissue vs.peripheral bloodN vs.B found a high-frequency mutant gene:PANK2(12%)?4.ConclusionIt is more suitable to use peripheral blood as normal control for gastric cardia adenocarcinoma than paracancerous tissue.