Detection Of Mouse Homologous Recombination Events During Meiosis Based On NGS Technology

The homologous recombination of mammalian germ cells occurs at the first meiotic prophase.Crossover between non-sister chromatid is an important reason for genetic diversity.Homologous recombination can be divided into two cases: one is bidirectional exchange of DNA molecules between two homologous chromosomes,which is called crossover;the other is one-way transfer of DNA molecules from one homologous chromosome to another homologous chromosome,which is called gene conversion.If there is a problem in the process of homologous recombination,it will lead to the corresponding diseases of the offspring.For example,the trisomy syndrome appeared in the population is due to the problem of homologous recombination in meiosis,which ultimately leads to the no separation of intermediates.This shows that the homologous recombination is closely related to the health of each of us,and it is necessary to study the homologous recombination in depth.But the present studies of meiotic homologous recombination are lack of effective experimental methods.The recombination rate calculated by statistical methods,need to establish on large population genetic data.Generally,we can only get recombination rates on large scale,and in the fine scale recombination rates cannot be calculated by this method.We hope that we can identified homologous recombination events on high resolution through the DNA sequencing technology,but the next generation sequencing have short reads,among the sequencer of the most popular company Illumina,Miseq series can sequencing longest read,but only can achieve the pair-end 300 bp and Miseq series sequencers belongs to the desktop sequencer.Compared with Hiseq series sequencers,Miseq Series sequencers have low throughput,high cost of sequencing and low accuracy.Hiseq sequencers can only get pair-end 150 bp read.This length of reads is not enough to identify homologous recombination events.The most representative technology of the third generation sequencing technology,is SMRT technology of Pac Bio company,it can get reads of tens KB to hundreds KB,the length is very long,but the single base error rate is very high,in order to obtain high accuracy data people need to increase the sequencing depth,it will be a sharp rise on the cost and the third generation sequencing technology overall is not mature enough,not as the stable as next generation sequencing system,downstream analysis technologies are not mature enough.So we use Illumina Tru Seq synthetic long read library build kit building the heterozygous mouse sperm DNA long reads library,after sequencing the library by Hiseq2500 sequencer of Illumina and then analyzed by bioinformatics,we identified the homologous recombination events that among the whole mouse sperm genome in the fine scale,then using heterozygous mice sperm DNA as materials to get the common whole genome sequencing data,analyzed the data we identified potentially recombination fragments,then we compared homologous recombination events and random fragments and found the overlap between recombination events and potential recombination fragments was significantly greater than the overlap between random fragments and potential recombinant fragments,suggest that homologous recombination events that we identified have high credibility,this method is useful.Previous studies have been reported,that PRDM9 histone methyltransferase is related to the formation of double strand break before homologous recombination.Using MEME motif finding software we found that PRDM9 motif is enriched in homologous recombination events.By located the homologous recombination events on to the genome function regions,we found that homologous recombination events are less in exons and introns,while there are more regions in intergenes,upstream and downstream of genes etc.The homologous recombination event tends to occur in the non functional regions or we can say in the less functional regions,which is consistent with the report of previous studies.Conclusion: We built heterozygous mouse sperm DNA libraries through Illumina Tru Seq synthetic long read library build kit,and then sequencing libraries through the next generation sequencing technology and obtained the whole genomic DNA sequencing data with 10 kb length,and then we identified the high resolution of homologous recombination events in fine scale(20bp ~ 5Kb)among whole genome,and proved the accuracy of the results.Further studies on these homologous recombination events have revealed that they tend to occur in regions with less functions,such as intergenes,upstreams and downstreams of genes,and avoid to occur in important functional regions such as exons and introns.