Table 4.

Challenges in microbiome forensics [58].

Challenge	Considerations
Microbiome Transfer	-The human microbiome can be transferred between cohabitants, pets or unknown people by physical interaction between them. -The human microbiome can be deposited into built environments. -The persistence of the microbiome on various surfaces is not well studied.
Sample collection	-Forensic examiners, protective clothing or tools can introduce a foreign microbiome. -Evidentiary items have the potential to transfer the microbiome to forensic examiners or the laboratory. -Environmental changes affect the evidence microbiome, which complicates sample storage. -Laboratory background microbial DNA needs to be continuously monitored.
DNA extraction	-Difficulties in reproducing a sample profile. -Extraction kits contain a background microbiome (kitome). -Samples can be outcompeted by contaminating microbial DNA.
Sequencing and analysis	-Microbial contamination can take place during sequencing. -Lowtemplate microbial DNA samples. -Indexhopping (reads assign to the wrong sample) and batch effects (unwanted variations introduced by confounding unrelated factors). -Bioinformatics are constantly evolving and cases must be revised with the new information.
Training and interpretation	-Methods and protocols are not validated. -Proficiency tests need to be developed. -There are no established forensic databases. -Likelihood ratio (LR) calculation needs development. -Mixture of microbiome profiles. -Bioinformatics tools’ complexity.