A Study Of Differential DNA Methylation Profile Between Two Monozygotic Twins

Objective: DNA methylation refers to the DNA of vertebrate animals is covalently modified by methylation at the cytosine base, which always occurs in the dinucleotide sequence 5'CpG3'. As a main branch of epigenetics, DNA methylation plays an important role in cell differentiation. Monozygotic (MZ) twins are always regarded'identical'in genetics since they arise from the same fertilized eggs. In the fields of forensic genetics, the current DNA analysis system can identify persons all over the world theoretically except MZ twins. So far, many studies on epigenetics of MZ twins have confirmed that there are many differences in the whole epigenome and specific regions, and epigenetic pattern is changing in a dynamic state throughout one's life. In this study we use methylated DNA immunoprecipitation sequencing(MeDIP-seq) to investigate and compare the DNA methylation profile of a pair of MZ twins, hoping to discover some different regions for forensic analysis.Method: Genome DNA was extracted from cord blood of 5 pairs of twins using DP-318 genome DNA extraction kit to investigate their short tamdem repeat(STR) with Identifiler? kit. Genome DNA from a pair of confirmed MZ twins was subjected to DNA methylation analysis with MeDIP-seq. The genome DNA was sonicated to random fragments ranging in size from 100 to 500 bp. End-repair of DNA fragment, addition of an adenine to the 3'end of DNA fragment, adaptor ligation were performed then. Denatured methylated single-strand DNA was captured by 5-methyl binding protein and specific regions were amplified to comfirm that the MeDIP specifically enriched methylated DNA and efficiently removed unmethylated DNA with qPCR. Enriched methylated DNA was purified and amplified to clone fragments of 200-300bp and sequenced with solexa. The sequencing reads were mapped onto the human genome reference, and the unambiguous reads were used to the following analysis including reads distribution, peak analysis, peak difference analysis and peak GO analysis. At last some different DNA methylation regions for forensic genetics were picked due to the result. Result:1 Basic biological information analysis: After (Acording to) the MeDIP-seq results, we obtained 3.6Gb data respectively, which contained 73 million reads for each sample. After mapped onto the human genome reference, we obtained 65 million reads respectively, occupying 88.75% and 89.26%. The number of unambiguous reads is 48 million and 50 million, occupying 74.77% and 76.35 in the mapped reads. The reads covers 33.25% and 38.76% of the genome with 3.22 and 2.76 times depth. Investigating the distribution of the unambiguous reads in the gene regions, we found that they appeared in every gene region but the maximum number of reads was in repeat elements. And then we investigated the distribution the unambiguous reads in the repeat element, finding that they also appeared in every element but the most were in the SINE/Alu sequence.2 Information for enrichment region of MeDIP-seq:We also investigated the peak information, obtaining 257 thousand and 197 thousand peaks for samle 1 and sample 2 respectively, covered 6.53% and 5.29% in the genome. After the length of the peaks were divided into 21 terms for 100bp each region, we found that the two samples had a similar distribution and in the region of 600-700bp they got a maximum number of peaks. After calculating the distribution of peaks in the gene regions, we found that these peaks had a widely distribution among gene regions but had a maximum number in the internal introns. These peaks also subjected to the Gene Oncology-based analysis. The identified GO categories had an unbalance distribution.3 Analysis of different DNA methylation regions:After analyzing the defferent regions between the two samples, we found 477 different gene related regions and got the same trend with fore-result from GO analsis. Totally, we found 2253 different DNA methylation regions through whole genome, 643 located in the gene regions and 1610 in the internal gene regions. From all of the regions, we finally selected 113 regions as markers for following forensic study.Conclusion:1 This study first profiled DNA methylation of a pair of MZ twins with MeDIP-seq, and totally found 2253 different DNA methylation regions through whole genome, 643 in the gene regions and 1610 in the internal gene regions.2 This study showed probability of DNA methylation in identifying MZ twins and finally selected 113 different DNA methylation regions for further study for the forensic application.