Analysis Of Time Since Deposition Based On MRNA Degradation And Microbial Variation Rules Of Biological Stains

Biological stains are one of the most common types of biological evidence at crime scenes and also the main objects of forensic identification providing important evidence for the individual and paternity identification related to case investigations.The time since deposition(TSD)of biological stains has played an important role in the inference of the time of crime,the identification of criminal suspects and the reconstruction of crime process,and has become a hot research topic in forensic science.However,no effective TSD inference methods have been established despite extensive attempts in forensic science and estimating TSD with reasonable precision still remains a challenge.In recent years,numerous studies confirmed that the degradation rule of biological stains could provide clues for the establishment of TSD analysis methods.Focused on the important scientific value of the degradation rule as well as the important role of TSD analysis in case investigation and court trial,blood and salivary stains,the most common biological stains with high DNA STR genotyping acquisition ratio were selected as the research objects.We analyzed the changes of RNA integrity(RIN)and transcript loss in blood samples and the characteristics of microbial diversity changes in salivary stains,proposed potential time-marker transcripts and microbes and established a TSD analysis method for blood samples and salivary stains.The main research contents and conclusions are as follows:(i)Blood samples from 20 volunteers were collected and prepared at a constant temperature of 4℃for 0–196 days.RNA sequencing was performed to investigate the rule of RNA degradation and transcript changes.The results showed that there was a significant temporal correlation in RNA integrity reduction and transcript loss(Pearson correlation coefficient r=-0.8672,p<0.001).During 0-168 days,RNA degradation and transcription loss in blood samples could be divided into four stages,i.e.,0–2 days(no significant change stage)(p>0.05),2–14 days(initial change stage)(p<0.001),14–56 days(rapid change stage)(p<0.0001),and 56–168 days(level out stage)(p>0.05).The blood samples of these four stages could be well distinguished in multidimensional scaling analysis(MDS),principal component analysis(PCA)and random forest analysis(area under the curve[AUC]=99.50%,precision=1,recall=1).FPKM(expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced)of transcripts in blood samples could be used for TSD analysis.(ii)Salivary stains from 50 volunteers were collected and prepared under constant temperature and humidity at 25℃for 0–196 days.16s amplicon sequencing was performed to investigate the characteristics of microbial community changes.The results showed that the number of ASVs(amplicon sequence variants),observed features,chao1 index,shannon index,simpson index and Pielou’s evenness index in salivary stains wavelike increased over time(p<0.05,R~2>0.8).The relative abundance of the dominant microbes Actinobacteriota,Proteobacteria,Fusobacteriota,Bacteroidota and Firmicutes were changed significantly over time(p<0.05)and the variation trend could be characterized by simple moving average.The relative abundance of microbial communities in salivary stains could be used for TSD analysis.(iii)While investigating the correlation between biostains degradation and time,variables including donor gender,carrier type and environment were set up to compare the effects of different factors on biostains degradation.The results showed that the TSD was the main factor for degradation while the donor gender did not significantly affect the changes of the transcripts of blood samples.Carrier types and time were the main factors for microbial changes in salivary stains.Under experimental conditions,different environments(room temperature environment vs.constant temperature and humidity environment at 25℃)and donor genders did not significantly affect the changes of the microbial species diversity and relative abundance of dominant species in oral swabs over time.TSD was the main factor for microbial community changes.(iv)Based on the random forest model,a method for TSD analysis of blood samples and salivary stains was established according to the FPKM of transcripts in blood sample and the relative abundance of time-related ASVs in salivary stains.The results showed that the blood samples or salivary stains in different time periods or different time groups could be well distinguished with sufficient theoretical basis.The method established in this research could be used for TSD analysis of biological stains.All of the prediction results of the same batch of test set samples were consist with the known TSD.The prediction accuracy of independent prepared blood samples was 47.22%,and the prediction results with positive reference value accounted for 77.77%.The prediction accuracy of independent prepared salivary samples was 59.87%,and the prediction accuracy of the samples with the known TSD of 0,3,14,21,84 and 112 days was higher than 80%.(v)One-way analysis of variance(ANOVA),kruskal-wallis test,linear discriminant analysis effect size(LEf Se)analysis,partial least squares discrimination analysis(PLS-DA),random forest analysis and KEGG(Kyoto encyclopedia of genes and genomes)functional pathway enrichment analysis were conducted to develop potential time markers.The results showed that 24 transcripts(MT-RNR1,etc.)and 25 microbes(Actinomyces,etc.)could be used as molecular markers for rapid analysis of TSD in case investigations.In summary,in view of the current difficulties in TSD analysis of biological stains in the field of forensic science,this study preliminarily established TSD analysis models for blood and saliva stains and carried out method testing on the basis of confirming that the rules of m RNA degradation and transcript loss in blood samples and the rules of microbial community changes in saliva stains were highly time-related.This research provides experimental basis and technical support for in-depth excavation of biological evidence,enhancement of criminal technical service capacity,and promotion of technological innovation of forensic science as well as a technical basis for the TSD analysis of biological stains for criminal case investigation.