Phenotype predictions of two-person mixture using single cell analysis.

Area of Science:

• Forensic genetics
• Genomic analysis
• Biotechnology

Background:

• Forensic DNA phenotyping (FDP) aims to predict externally visible characteristics from DNA evidence.
• Implementation of FDP in routine casework faces challenges, particularly with DNA mixtures.
• Accurate phenotype prediction from mixed DNA samples is crucial for advancing forensic investigations.

Purpose of the Study:

• To evaluate single-cell sequencing as a method for reliable DNA-based appearance prediction from DNA mixtures.
• To assess the efficacy of combining single-cell separation with targeted sequencing panels for mixture deconvolution.
• To determine if this approach can enable accurate phenotyping of individual contributors within forensic samples.

Main Methods:

• Analysis of two mock DNA mixtures (two contributors each) using two distinct workflows.
• Workflow 1: Direct sequencing of mixtures with the Ion AmpliSeq™ PhenoTrivium Panel (41 HIrisPlex-S markers) followed by HPS Deconvolution Tool analysis.
• Workflow 2: Single-cell separation and collection using the DEPArray™ PLUS System, followed by PhenoTrivium amplification and phenotype prediction, compared against HPS Tool results.

Main Results:

• Single-cell sequencing enabled nearly complete HIrisPlex-S profiles with accurate genotypes.
• Reliable phenotype predictions were achieved for individual contributors within the analyzed mixtures.
• The presented method successfully deconvoluted mixtures, demonstrating its potential for forensic applications.

Conclusions:

• Single-cell sequencing combined with targeted panels offers a robust solution for DNA mixture deconvolution in forensic DNA phenotyping.
• This approach overcomes significant obstacles in routine FDP, enabling accurate appearance predictions from complex samples.
• The methodology holds promise for enhancing the capabilities of forensic investigations through advanced DNA analysis techniques.