Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing

Abstract

The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable 'source level' information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and inter-sample differential gene expression revealed by agglomerative hierarchical clustering.

The stability and persistence of blood and semen mRNA and miRNA targets for body fluid identification in environmentally challenged and laundered samples

“…The transcripts also persisted through wash cycles and machine drying. Implementation of stable region transcripts (STaRs) [45] or the use of massively parallel sequencing [46] may further improve body fluid identification analyses for challenging forensic samples. Fig.…”

Body fluid identification using a targeted mRNA massively parallel sequencing approach – results of a EUROFORGEN/EDNAP collaborative exercise

“…In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the MiSeq/FGx MPS platform [27]. To encourage other laboratories not having access to a MiSeq/FGx platform to participate, an ad hoc in-house PGM/S5 assay was developed for this study.…”

“…MiSeq/FGx laboratories used a TruSeq Targeted RNA Expression panel (Illumina Inc., San Diego, CA) to amplify the 33 body fluid specific transcripts [27] (Table 1). PGM/S5 laboratories used an Ion AmpliSeq™ RNA custom panel (Thermo Fisher Scientific) detecting 29 body fluid specific transcripts (Table 1).…”

“…Furthermore, MPS is specifically suitable for degraded samples, since mRNA is usually fragmented prior to the reverse transcription step in MPS protocols. Within the EUROFORGEN Network of Excellence (https://www.euroforgen.eu) we established a targeted mRNA massively parallel sequencing approach for body fluid/tissue identification [27]. This included the evaluation of a suitable library preparation protocol and sequencing platform, as well as the selection of the most appropriate markers, based on the experience with the PCR/CE-system.…”

“…semen, saliva, vaginal secretion, menstrual blood and skin. In a proof-of-conceptpaper the assay we developed and ran on the Illumina MiSeq/FGx platform was demonstrated to identify body fluids with good sensitivity in single source and mixed stains [27].…”