Table 1.

Summary of the potential utility and advantages of microbial profiling, with associated limitations and challenges associated with using the human microbiome for forensic human identification and/or association via a 16S rRNA bacterial profiling approach.

Potential Utility/Advantages	Associated Limitations and Challenges
▪Human microbiomes may be highly individualised. ▪Human microbial profiling may assist investigation of criminal activities by providing information not available utilising current forensic methodologies.	▪Human microbiomes can be transferred between people, both directly and indirectly. ▪Unlike human DNA, human microbiomes differ among areas of a body, change over an individual’s lifetime, and are dependent on habits and environments occupied. ▪Human DNA may degrade but its profile does not change when a forensically relevant sample is collected sometime after it has been deposited, while the human microbial profile may change over this period. ▪Forensic human DNA profiles are made up of core sets of loci with alleles of known population frequency, while human microbial profile components are plentiful, variable, and their frequency and proportions within sample types are relatively unknown.
▪Human-associated microbiota are in high abundance compared to human somatic cells, and may be more readily available from crime scenes than human DNA. ▪Microbial profiles can be produced from low-biomass samples, which may be useful for trace evidence.	▪A sample of almost anything (e.g., surfaces, items, equipment, reagents) can produce a microbial profile, potentially compromising the interpretability of the microbial profile of the target sample. ▪As human microbiomes can be altered or change over time, the lapse in time between microbial sample collection from a crime scene and microbial sample collection from potential suspect(s) may impact comparisons. ▪It must be demonstrated that microbial profiles of low-biomass samples are different from profiles of control samples. Negative extraction controls often return non-negative results and low-biomass samples are often impacted by exogenous microbial contamination.
▪Human-associated microbial communities are driven by body-site differences.	▪Identifying body sites associated with a sample may only be useful for investigations in which potential suspect(s) have been identified. ▪If no suspect(s) have been identified, it may be challenging to determine if a microbial sample, and the associated profile, is of mixed-origin.
	▪Microbial contamination can occur throughout sample collection to sequencing and may originate from multiple technical and environmental sources.
	▪Microbial forensics may require new standard operating procedures to collect, store and extract microbiome samples. These new procedures may not be compatible with existing procedures designed and optimised for human nDNA recovery.
	▪Microbial profiles may be difficult to reproduce, especially if a different collection protocol, DNA extraction kit, technician or environment (i.e., forensic laboratory setting) is applied.
	▪Current recommendations for microbiome studies to reduce variation, including sequencing batch effects, may not be suitable for forensic protocol, and for microbial profiling of evidentiary and questioned samples.
	▪Bioinformatics is constantly evolving, which may influence the outcome of microbial profiles and thus hinder valid comparisons of profiles determined by different tools.
	▪Forensic practitioners are by and large unfamiliar with the methodologies and procedures of generating, interpreting and reporting microbial profiles, thus a significant investment in training and authorisation would be required.