TrACES of time: Towards estimating time-of-day of bloodstain deposition by targeted RNA sequencing

In forensic molecular biology, the main task consists of identifying individuals who contributed to biological traces recovered from (potential) crime scenes. However, to support evidence-based reconstruction of the course of activities having taken place at the scene, contextualising information regarding how and when a biological trace was deposited is oftentimes required. Here we present the development of a forensic molecular biological analysis procedure for the prediction of the time-of-day at which a bloodstain has been deposited by targeted quantification of selected mRNA markers. Time-of-day candidate prediction markers with diurnally rhythmic expression have previously been identified by whole transcriptome sequencing. Here, we build on our previous findings by establishing a targeted cDNA sequencing protocol on an Ion S5 massively parallel sequencing device for the targeted gene expression quantification of 69 time-of-day candidate prediction markers. Based on expression measurements of these markers in 408 blood samples (from 51 individuals deposited at eight time points over a day), we establish and compare different statistical methods to predict time of deposition. The most suitable model employing penalised regression achieved a root mean squared error of 3 h and 44 min with 78 % of predictions being correct within ± 4 h (evaluated by five-fold cross-validation), showing pronounced inter-individual differences. While the prediction accuracies of the method in its current state limit its use in the evaluative stage of a criminal trial, the method may nonetheless provide valuable information in the investigative phase. Our study provides the first prediction model for time-of-day of bloodstain deposition based on targeted RNA sequencing and thus represents an important step towards forensic trace deposition timing. It thereby relevantly contributes to the growing knowledge on Transcriptomic Analyses for the Contextualisation of Evidential Stains (TrACES).