Pushing the Boundaries: Forensic DNA Phenotyping Challenged by Single-Cell Sequencing.

Marta Diepenbroek,Birgit Bayer,Katja Anslinger

doi:10.3390/genes12091362

Marta Diepenbroek, Birgit Bayer + Show 1 more

Open Access

https://doi.org/10.3390/genes12091362

Copy DOI

Journal: Genes	Publication Date: Aug 30, 2021
Citations: 11	License type: CC BY 4.0

Affiliation: Ludwig-Maximilians-Universität München

Abstract

Single-cell sequencing is a fast developing and very promising field; however, it is not commonly used in forensics. The main motivation behind introducing this technology into forensics is to improve mixture deconvolution, especially when a trace consists of the same cell type. Successful studies demonstrate the ability to analyze a mixture by separating single cells and obtaining CE-based STR profiles. This indicates a potential use of the method in other forensic investigations, like forensic DNA phenotyping, in which using mixed traces is not fully recommended. For this study, we collected single-source autopsy blood from which the white cells were first stained and later separated with the DEPArray™ N×T System. Groups of 20, 10, and 5 cells, as well as 20 single cells, were collected and submitted for DNA extraction. Libraries were prepared using the Ion AmpliSeq™ PhenoTrivium Panel, which includes both phenotype (HIrisPlex-S: eye, hair, and skin color) and ancestry-associated SNP-markers. Prior to sequencing, half of the single-cell-based libraries were additionally amplified and purified in order to improve the library concentrations. Ancestry and phenotype analysis resulted in nearly full consensus profiles resulting in correct predictions not only for the cells groups but also for the ten re-amplified single-cell libraries. Our results suggest that sequencing of single cells can be a promising tool used to deconvolute mixed traces submitted for forensic DNA phenotyping.

Highlights

A single human cell contains around six pg of DNA, which is translated to approximately 2 × 3.3 billion base pairs
In order to conduct cell separation, the biological material was first stained with the DEPArrayTM Forensic SamplePrep Kit (Menarini Silicon Biosystems, Bologna, Italy), which enables the staining of epithelial cells, leucocytes, and sperm cells
The study consisted of 5 groups of 20 cells, 9 groups of 10 cells, and 10 groups of 5 cells, for a pool of 24 libraries total, which was sequenced on a 530 Chip

Summary

Introduction

A single human cell contains around six pg of DNA, which is translated to approximately 2 × 3.3 billion base pairs. Sequencing of this amount of genomic data became possible thanks to the rapid development of next-generation sequencing (NGS) [1]. The efficacy of single-cell sequencing in medicine cannot be overestimated, and it demonstrates the possibility for implementation in forensics, where it is not commonly used. The main motivation behind introducing this technology into forensics is the potential to improve mixture deconvolution workflows and interpretation methods. Implementing single-cell analyses can improve the efficacy of mixture deconvolution and, has already gained interest in the forensic community. A cell separation solution is the DEPArrayTM N×T System from Menarini Silicon

Methods

Results

Discussion

Conclusion