Preliminary Study On Three-dimensional Structure Of Translocation Chromosomes Based On Hi-C Technique

Chromosomal abnormalities can cause structural or functional abnormalities in male and female sex organs,which is one of the important causes of poor motherhood or infertility.Statistics show that about 10-15%of married couples suffer from infertility.One of the main reasons is the abnormal chromosome number or structure.Chromosomal translocation is one of the common causes of abnormal chromosome structure and is one of the important causes of female spontaneous abortion,fetal malformation or fetal chromosomal abnormalities~].With the unprecedented development of science and technology,more and more studies have shown that the spatial organization of eukaryotic chromatin in the nucleus has an important influence on the spatial distribution of DNA,and thus regulates biological processes such as DNA replication and gene transcription.The current understanding of this highly spatially ordered genome structure is still rough,fragmented,and incomplete.In recent years,with the development of linear genomes,researchers have used fluorescence in situ hybridization,chromatographic conformation capture(3C)~]and other technologies to gain a deeper understanding of the three-dimensional conformation and content of chromatin in the nucleus.Hi-C(highest-throughput chromosome conformation capture),a derivative technique developed using the chromatin conformation capture technique,is an analytical technique for studying genome-wide chromatin interactions and exploring the three-dimensional structure of whole genomes.Although Hi-C technology is derived from 3C,the entire operation process is more complicated than that of 3C,and there are many reagents and equipment involved in different enzymes.Therefore,this study validates the reliability of the conventional Hi-C process according to the current Hi-C method,which is currently cumbersome and not easy to perform quality control.The use of Hi-C technology enables a fine analysis of the interactions of chromatin or all chromatins,then bringing gene expression regulation to a spatial,global level of research for the comprehensive resolution of DNA-related biological processes.The mechanism opens up new opportunities.Objective:A conventional Hi-C library was established to verify the reliability of Hi-C process repeatability,and the key steps and precautions were noted to provide reference for future researchers.In combination with bioinformatics analysis technology,Hi-C data and corresponding heat maps were analyzed to explore the spatial three-dimensional structure of translocation chromatin at the level of the whole genome.The main purpose of this study is to explore the spatial three-dimensional structure and its correlation with pathogenicity,provide a research direction and theoretical basis for adverse pregnancy outcomes caused by chromosomal abnormalities,and it is of great significance to guide fertility.Methods:In this study,the patient was a patient from the 181th Hospital of the Chinese People's Liberation Army who performed peripheral blood karyotype analysis.A patient with a chromosome karyotype translocation and a healthy person with normal karyotype were selected and their peripheral blood was collected.After whole-genome Hi-C sequencing was performed in the case group and the control group,preliminary bioinformatics analysis of high-throughput genome sequencing was performed on the raw data obtained from the two groups.Bioinformatics analysis begins with the acquisition of sequencing raw data.First,in the quality control phase,the linker sequence,the contaminated,low-mass sequence of the linker are removed and"clean data"is obtained.The follow-up analysis is based on filtered data analysis.Then use the HiC-Pro's comparison strategy,call bowtie2 to compare,and then we can get the Reads logarithm to the unique position of the genome.Finally,Hic-Pro is used to analyze the Hi-C data to obtain a corresponding matrix heat map.Results:(1)In this study,four Hi-C libraries were constructed,and the results of the quality control points of the conventional Hi-C process all met the Hi-C database requirements.(2)The case and control groups generated approximately 270 Gb-scale sequences for the original Reads of 947,652,970 and 913,192,394,00,respectively,and achieved very high coverage of 94.72%and 94.95%by sequencing at 100 kb.(3)The cis-acting Reads obtained in the case and control groups were 237,062,419 and266,285,718;Reads for trans interactions are 94,115,136 and 146,084,685,respectively.(4)This study obtained the ratio of cis-interaction and trans-interaction at the genome-wide level.(5)The whole genome interaction heat map between the case group and the control group was obtained.(6)The genome-wide heat maps of the case group and the control group suggested that chromosome 1 and X interactions were enhanced after translocation.Conclusion:(1)Through the conventional Hi-C method and the quality control during the entire construction process,the four Hi-C libraries established in this study are all in compliance with the requirements,confirming the reliability of the conventional Hi-C process.(2)In this study,the preliminary biological information analysis was performed on the data obtained from Hi-C sequencing and screened out for efficient sequencing of Reads.The ratio of cis-trans interactions between the case group and the control group is known to be greater than the cis-acting interaction,which has a stable cell type or species.(3)This study uses Hi-C analysis software Hic-Pro analysis of Hi-C data to obtain the mutual interaction of chromosomes.It is known that the intra-chromosomal interaction is stronger than the interaction between chromosomes.(4)On the basis of the inter-chromosomal interaction heat map,the y-axis dimension of the heat map matrix is sorted according to the chromosome size,and the X-axis dimension is clustered according to the interaction value between chromosomes.It can be seen that some small genes are rich in genes.Chromosomes preferentially interact.(5)Comparison of whole-genome heat maps between the case group and the control group revealed that chromosome 1 and chromosome X in the case group interacted more strongly than the control group.It may be that after the translocation of chromosomes 1 and X,the spatial distance became smaller,resulting in mutual With the enhanced effect,it can be inferred that the chromosomes with closer spatial distances interact more strongly.