Single-cell deoxyribonucleic acid typing for forensic mixtures and trace evidence: Opportunities, validation requirements, and reporting limits

Table 1 Proposed validation and implementation checklist for single-cell and single-molecule analysis for deoxyribonucleic acid identification-like forensic workflows

Validation domain	Proposed minimum deliverable or benchmark for intended use
Cell recovery and sampling	Report the probability of recovering at least one target minor-contributor cell under casework-like conditions; if the workflow is used to support absence-sensitive reasoning, the validated capture probability for the claimed scenario should be explicitly stated
Cell-type classification	Publish sensitivity, specificity, positive predictive value, and misclassification rates by substrate, staining condition, and sample age; do not use phenotype labels to support source-level wording without validated error rates
Minimum pooling and consensus	Validate the minimum number of cells needed to reach the laboratory’s pre-specified completeness target; current evidence supports at least small diploid pools, and approximately 15 sperm cells to exceed 90% inferred autosomal diploid completeness in one validated DEPArray setting
Dropout and stutter	Publish locus-specific dropout and stutter distributions by cell type and workflow; define no-call rules for loci that fail consensus or replicate criteria
Contamination and drop-in	Quantify stage-specific unexpected-allele and drop-in rates; require no interpretable human profile in reagent blanks for routine release and pre-define batch action limits for recurrent exogenous signal
Reproducibility and robustness	Demonstrate reproducibility across operators, instruments, days, substrates, and biologically realistic mixtures; include degraded, aged, and background-rich samples rather than only clean artificial mixtures
Concordance with reference methods	Demonstrate concordance of recovered contributor genotypes against known reference samples and against bulk STR where comparison is informative
Database compatibility	Validate upload eligibility separately from evidential usefulness; document whether resulting profiles meet local locus-count, allele-count, and statistical requirements for database searching
Reporting boundaries	Pre-define permissible outputs at sub-source and limited source level; state that cellular resolution does not by itself resolve transfer, persistence, prevalence, recovery, or timing

STR: Short tandem repeat.