# Spitting in the wind?—The challenges of RNA sequencing for biomarker discovery from saliva

## TL;DR

This paper explores the challenges of using RNA sequencing to find biomarkers in saliva for forensic analysis, highlighting issues like bacterial contamination and RNA degradation.

## Contribution

The paper provides insights and recommendations for improving RNA biomarker discovery in saliva for forensic trace contextualization.

## Key findings

- RNA sequencing of saliva is complicated by oral bacteria and sample heterogeneity.
- Degradation of RNA in saliva samples poses a significant challenge for biomarker discovery.
- Recommendations are proposed to enhance RNA biomarker identification in saliva.

## Abstract

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA (“RNA sequencing”) has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

The online version contains supplementary material available at 10.1007/s00414-023-03100-3.