A Study On Whole Genome Sequencing Identifies Nucleosome Protected Loci And Its Forensic Application

Objective:To establish a multiplexed SNPs amplification system based on the massive parallel sequencing platform,which was located on nucleosome histone binding region and applied for degraded samples.Because of the protection of histone octamer,DNA twisted in this region was difficult to degradation.In this study,the SNPs located on the nucleosome histone protecting region were chosen to establish a multiplex panel on NGS platform,which was appropriate for the degradative samples' detection.Methods:Micrococcus nuclease(specific digest the nucleosome junction)was used to digest the DNA isolated from different human tissue samples,after library preparation and quantification,all the samples were sequenced on the HiSeq 2000 sequencing platform.Bioinformatics methods as mapping and comparative were used to screen markers following two basic principles: showed well frequency data in Chinese population and easy to composite with each other.Through primers design,multiplex system establishment,all the chosen SNPs were detected in one experiment.The sensitivity,repeatability and concordance of the system were also evaluated with standard template DNA.We finally tested the frequencies of this markers in Chinese Han population due to the further application of this system in forensic practice.Results:In total,11,897,115 SNP were found in the DNA sequences of nucleosome histone protecting region originated from lung,liver,spleen and muscle.Among them,226,395 SNP sites met the requirements of polymorphism frequency close to 0.5.Finally,the multiplex assay including 40 SNP loci was established based on the massive parallel sequencing platform.The system showed high sensitivity of 1ng input DNA,even with 0.1ng input DNA,90% markers were detected successful.For the highly degradative sample(free blood DNA),it showed the precision rate of 95%.Conclusion:Based on the characteristic of nucleosome twisted DNA protected by the histone,we established a multiplex panel of 40 SNP to appeal the application for degradative samples.In the evaluation experiments,the system presented high sensitivity,well repeatability and concordance,which would show a great advantage in the application for degradative samples.Our study presented a new approach and created a new method of degradative samples' detection in forensic practice.