Microbiome-based body site of origin classification of forensically relevant blood traces

Human blood traces are amongst the most commonly encountered biological stains collected at crime scenes. Identifying the body site of origin of a forensic blood trace can provide crucial information in many cases, such as in sexual and violent assaults. However, means for reliably and accurately identifying from which body site a forensic blood trace originated are missing, but would be highly valuable in crime scene investigations. With this study, we introduce a taxonomy-independent deep neural network approach based on massively parallel microbiome sequencing, which delivers accurate body site of origin classification of forensically-relevant blood samples, such as menstrual, nasal, fingerprick, and venous blood. A total of 50 deep neural networks were trained using a large 16S rRNA gene sequencing dataset from 773 reference samples, including 220 female urogenital tract, 190 nasal cavity, 213 skin, and 150 venous blood samples. Validation was performed with de-novo generated 16S rRNA gene massively parallel sequencing (MPS) data from 94 blood test samples of four different body sites, and achieved high classification accuracy with AUC values at 0.992 for menstrual blood (N = 23), 0.978 for nasal blood (N = 16), 0.978 for fingerprick blood (N = 30), and 0.990 for venous blood (N = 25). The obtained highly accurate classification of menstrual blood was independent of the day of the menses, as established in additional 86 menstrual blood test samples. Accurate body site of origin classification was also revealed for 45 fresh and aged mock casework blood samples from all four body sites. Our novel microbiome approach works based on the assumption that a sample is from blood, as can be obtained in forensic practise from prior presumptive blood testing, and provides accurate information on the specific body source of blood, with high potentials for future forensic applications.