Exploration Of The Next-Generation Sequencing-Based Integrated Detection Technology For Forensic Deoxyribonucleic Acid Genetic Markers And Ribonucleic Acid Molecular Markers

Objectives:Deoxyribonucleic acid（DNA）genetic markers and ribonucleic acid（RNA）molecular markers have been widely used for forensic practices.DNA genetic markers can be used for individual identification,parentage testing,and biogeographic ancestry inference.RNA molecular markers can be used for body fluid identification,determination of the age of stains,and molecular pathological diagnosis.Currently,DNA and RNA are often studied separately.However,this research methodology has some disadvantages,including complicated experimental procedures,high sample consumption,and weak internal connectivity of biomarkers.Variant information from DNA/RNA markers and their interrelation could be revealed by a single-stream workflow.Integrated detection workflow aims to simplify working protocols,reduce time consumption and save valuable samples collected from crime scenes.Compared with the capillary electrophoresis technology commonly used in forensic laboratories,next-generation sequencing（NGS）technology characterized by high throughput,high speed,and high integration,can detect multiple types and a large number of biomarkers,which is more suitable for DNA and RNA integrated detection.Limited research on NGS-based DNA/RNA integrated detection technology has been reported at present,especially in forensic medicine.Therefore,the purpose of this study is to explore the technical method suitable for the integrated detection of forensic DNA genetic markers and RNA molecular markers on the NGS platform.Methods:1.Comparison of DNA and RNA co-extraction methodsThe QIAamp DNA Investigator Kit,Mag Attract M48 DNA Manual Kit,Prep Filer Express BTA^TMForensic DNA Extraction Kit,Ultrapure RNA Kit,miRNeasy Micro Kit,and All Prep DNA/RNA Micro Kit were used to extract peripheral blood samples from five individuals.The quantification results of DNA and RNA were compared.2.Comparison of nucleic acid quantification methodsA fluorometric measurement using a Qubit fluorometer and a photometric measurement using a Nano Drop spectrophotometer were used to quantify DNA and RNA in total nucleic acid.The quantification results were compared.3.Selection of DNA and RNA markers and panel design for the integrated detection panelDNA markers for individual identification were selected,including short tandem repeat（STR）and single nucleotide polymorphism（SNP）.Peripheral blood-specific and saliva-specific messenger ribonucleic acid（mRNA）markers were selected for body fluid identification.Primers for DNA and RNA markers were designed and verified.All selected markers were used to construct an integrated detection panel.4.Exploration of the impact of reverse transcription reaction on the detection of forensic DNACapillary electrophoresis-based and NGS-based methods were used to detect the DNA solution before and after reverse transcription.Obtained STR and SNP genotypes were compared,and the average relative fluorescent unit and depth of coverage in each sample were analyzed.5.Exploration of the method for co-preparation of DNA and RNA librariesTwo methods were used to simultaneously prepare DNA and RNA libraries.Method 1:The reverse transcription product of total nucleic acid was directly used to prepare DNA and RNA libraries.Method 2:The mixture of total nucleic acid and reverse transcription product of total nucleic acid was used to prepare DNA and RNA libraries.6.Sample detection and accuracy studyTen body fluid samples（five peripheral blood samples and five saliva samples）were detected by the integrated detection panel on a Miseq FGx machine.Run metrics were summarised.Several experiments were conducted to detect the accuracy of STR genotypes,SNP genotypes,and mRNA expression.Results:1.The Ultrapure RNA Kit and miRNeasy Micro Kit could be used to simultaneously extract DNA and RNA from all samples.However,although DNA could be extracted from all samples using the QIAamp DNA Investigator Kit,Mag Attract M48 DNA Manual Kit,Prep Filer Express BTA^TMForensic DNA Extraction Kit,and All Prep DNA/RNA Micro Kit,the results of RNA extraction is not ideal,and RNA could only be extracted from part of samples.2.The quantitative results from the Nano Drop spectrophotometer and the Qubit fluorometer were quite different for the quantification of the mixture of DNA and RNA.The Nano Drop DNA quantitative results were 1.2 to 7.2 times higher than the Qubit quantitative results.The Nano Drop RNA quantitative results were 2.1 to 14.6times higher than the Qubit quantitative results.3.Ten autosomal STR loci,45 autosomal SNP loci,and eight mRNA molecular markers were selected.Primers for these markers had good specificity.4.After reverse transcription,STR and SNP were detectable in the DNA solution,and the genotypes were identical to those of the DNA solution without reverse transcription.5.DNA and RNA libraries could be simultaneously detected using method 1 or method 2.6.STR genotypes,SNP genotypes,and mRNA expression could be simultaneously detected in ten body fluid samples using the DNA and RNA integrated detection panel..This panel’s high level of accuracy was confirmed.Conclusions:In this study,an NGS-based method for the integrated detection of forensic DNA genetic markers and RNA molecular markers was developed,and an integrated detection panel was designed,which could simultaneously detect ten autosomal STR loci,45 autosomal SNP loci,and eight mRNA molecular markers.The integrated detection panel could be used for simultaneous individual and body fluid identification on human peripheral blood or saliva samples.This study was the first in the world to realize the highly integrated detection of forensic DNA genetic markers and RNA molecular markers.Compared with other related studies,this method was highly integrated and more efficient,bringing a new pathway for realizing the full utilization of biological samples.