Establishment And Application Of A PCR-based Target Enrichment Sequencing Method Of HERV-K(HML-2)

Human endogenous retrovirus K(HERV-K)(HML-2),the most recent integration group with the least number of mutations and the most biologically active,has been reported to be reactivated in tumors and autoimmune diseases.Meanwhile,HERV-K(HML-2),is suspected to be transposition-defective,and may reshape the transcriptional network of the human genome by regulatory elements distributed in their long terminal repeats(LTRs).Insertionally polymorphic HERV-K(HML-2)loci,which have been identified with different prevalence in various kinds of diseases and different prevalence in ethnic populations are considered as one explanation of how ubiquitous elements such as HERV-K(HML-2)can cause disease in only a proportion of individuals.These elements are potential candidates for studying human evolution.Because of the high sequence similarity between different HERV-Ks,current methods have limitations in providing genome-wide mapping specific for individuals HERV-K(HML-2)members.This is a major barrier in delineating HERV-K(HML-2)function.In the present study,we are attempted to establish an effective method for completing the full genomic distribution of HERV-K(HML-2)in the human genome.Furthermore,we sought to identify polymorphic integration loci by comparing the relative abundance of the same loci in different individuals.In this study,a PCR-based target enrichment sequencing of HERV-K(HML-2)(PTESHK)has been established to amplify and determine individual HERV-K(HML-2)element insertion sites within human genomic DNA.PTESHK takes advantage of target enrichment amplification of HERV-K(HML-2)integration sites and construction of libraries for obtaining HERV-K(HML-2)individual proviruses or HERV-K(HML-2)solo-LTR sequences linked to insertion site sequences.PTESHK can map the presence of known loci and identify novel loci,enabling determination of the genome-wide distribution of HERV-K(HML-2)loci.Here we report on the genomic data obtained from three individuals(three replicates each).We identified a total of 978 loci using this method,including 35 new loci.Our ultimate purpose behind developing PTESHK in the identification of genome-wide distribution of HERV-K(HML-2)in the human genome was to distinguish polymorphic integrations among different individuals or cohorts.Among the 3 individuals in our study,14 polymorphic HERV-K(HML-2)loci were identified,and solo-LTR330 and N6p21.32 were identified as polymorphic for the first time.After the establishment of PTESHK,we used it to identify the distribution of HERV-K(HML-2)loci in four prostate cancer cell lines,including DU145,LNCa P,PC-3,and VCa P.Similarly,each cell line was performed in triplicate.According to the number of detected HERV-K(HML-2)loci of different replicates from the same prostate cancer cell line,PTESHK was further used to confirmed with good repeatability for detecting the genome-wide distribution of HERV-K(HML-2)loci.Among the 4 prostate cancer cell lines in this study,26 polymorphic HERV-K(HML-2)loci were identified,indicating PTESHK’s ability in detecting polymorphic integrated HERV-K(HML-2).Compared with cell lines DU145,LNCa P,and VCa P,PC-3 cell lines were absent in6 HERV-K(HML-2)loci located on chromosome Y.This is consistent with other studies showing no normal Y chromosomes detected in PC-3 cell lines.We speculate the identification and the distribution of HERV-K(HML-2)loci,especially distinguishing polymorphic integrated loci,can represent aberration of chromosomal elements,which may be used as a candidate biomarker for different types of prostate cancer.In the present study,PTESHK performed well in detecting polymorphic integrated HERV-K(HML-2)loci among samples with experimental replicates.To optimize PTESHK for samples without replicates,we organized data from the above three individuals and four cell lines to identify a list of HERV-K(HML-2)loci which can be detected by PTESHK with good repeatability or relatively higher CPM values.In the list,there are 638 HERV-K(HML-2)loci.To evaluate the capacity in detecting polymorphic integrated HERV-K(HML-2)loci with these loci,4 individuals were collected for construction of PTESHK libraries without experimental repeats.Among the 4 individuals,19 polymorphic loci were detected.Using specific primers for each polymorphic locus,14 of the 19 loci were verified by nested-PCR.The 5 exception loci may be caused by their location in a repeat element.Thus,PTESHK performs well in identifying polymorphic loci among samples with only 1experiment had been run.To determine whether HERV-K(HML-2)still mobilizable in human genome and to better understand the possible function of HERV-K(HML-2)during development,four pairs of monozygotic twins,including two pairs of newborns and two pairs of elders,were recruited to perform PTESHK for identifying de novo integrations.One different HERV-K(HML-2)locus was identified between one pair of newborn twins.Unfortunately,the locus failed to be verified by specific PCR.In the other three pairs of the monozygotic twins,no different HERV-K(HML-2)locus was identified between pair of twins.Therefore,we cannot conclude whether there is de novo integration or not,but it still provides evidence for further study on the possible mobility of HERV-K(HML-2).We performed PTESHK in 47 individuals from two different ethnic populations to identify polymorphic integrated HERV-K(HML-2),which may be helpful for understanding the genetic diversity among different populations.Among the two groups of individuals,36 polymorphic loci were identified.Interestingly,there are 8 loci that exhibited significant differences on prevalence between the two ethnic groups,which might contribute to the genetic diversity between Chinese and Italians.When comparing the prevalence of polymorphic integrated HERV-K(HML-2)loci among Europeans,Asians,and Africans,all HERV-K(HML-2)loci detected in Europeans and Asians could be validated in Africans,implying their insertion before human migration out of Africa.And a significant tendency for fixed(prevalence = 1)or unoccupied(prevalence = 0)of several loci were identified in Asians and Europeans compared with Africans,which represents a loss of genetic diversity.Therefore,the detection of HERV-K(HML-2)loci and their prevalence in different ethnic populations may offer researchers a new genomic avenue aid in the study of human migration and evolution.In general,we developed PCR-based target enrichment sequencing of HERV-K(HML-2),PTESHK,to assess the genome-wide distribution of HERV-K(HML-2)in human genome.In addition to the ability to characterize the distribution of HERV-K(HML-2)in the human genome,PTESHK performs well for the detection of polymorphic loci and de novo integrated HERV-K(HML-2)loci within different disease type and in different ethnic groups.This method could be a supplementary technique for understanding the physiological and pathological mechanisms of HERV-K(HML-2),and its potential functions in human evolution.