The Power of Single Cells in Forensics
Genotyping of DEPArray™ Isolated Single Cells with PowerPlex® Fusion 6C System
Francesca Fontana, R&D Biology Manager, Menarini Silicon Biosystems, Italy
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Introduction
The genetic investigation on crime scenes requires collection and analysis of biological evidence for DNA profiling. Body fluids left behind by perpetrators contain blood, sperm and epithelial cells that can help investigators acquire the genetic profile of people involved with the crime.
The problem occurs when evidence consists of mixed biological fluids from two or more persons (e.g. the victim and one or more perpetrators).
Biological mixtures can be those where i) each contributor is represented with a different type of body fluid/cell type (i.e. sperm-epithelial-cells, blood-epithelial cells, blood-sperm) and those where ii) more than one contributor is represented by the same body fluid/cell type (blood-blood, sperm-sperm, epithelial cells-epithelial cells).
Current approaches to resolve mixed biological evidence profiling include the chemical reduction of one type of cells (differential lysis) or the use of algorithms to statistically deconvolute complex DNA profiles. These methods are limited however to mixtures of sperm/epithelial cells and to a limited range of DNA ratios.
The value of single cell genotyping
In this context, genotyping single cells precisely isolated from the evidence could minimize the loss of critical information in complex mixtures and enable obtaining pure genetic profiles to unequivocally identify the contributors to the mixture. Different approaches have been applied to isolate single cells from biological mixtures such as Laser Capture Microdissection (LCM) and Fluorescence Activated Cell Sorting (FACS) but they failed in reaching throughput, sensitivity or purity needed (1).
The DEPArray™ technology allows to isolate pure single cells or small pools of cells from biological evidence through digital sorting, reaching single cell level of purity, thus potentially solving the riddle of complex mixtures (1–4).
Pure pools of cells might be then sufficient to resolve cases where victim and perpetrator contribute with a different cell type while, for more complex cases, single cell profiling is the ultimate solution to identify all contributors. Pure, DEPArray™ isolated single cells can be genotyped with standard kits providing sufficient sensitivity for low input DNA (1–4).
Nevertheless, DNA profiling of single cells offers a number of challenges due to the limited amount of gDNA available for analysis, which is estimated to be 6.6pg for diploid cells (e.g. white blood cells, vaginal and buccal epithelial cells) and 3.3pg for haploid cells (spermatozoa). Therefore, a performance evaluation is needed to adopt optimized protocols when performing single cell STR profiling.
Here we show the performance evaluation data obtained testing the PowerPlex® Fusion 6C System, that co-amplifies 27 total loci (23 autosomal STRs, 3 Y-STRs and Amelogenin), on multiple single cells, both diploid and haploid, isolated by DEPArray™ from a mixture composed of semen, blood and saliva from three different donors.
Figure 1: DEPArray PLUS workflow for single cell forensics
Figure 2: PowerPlex® Fusion 6C System Loci Configuration.
Identification and isolation of single cells
Samples were prepared using body fluids obtained upon donors’ informed consent. An aliquot of each biological fluid (semen, saliva, blood) used for the preparation of samples, was collected for gDNA extraction, in order to produce reference profiles for comparison purposes.
Three different donors were asked to provide a single type of body fluid each as described in Table 1.
Table 1: Biological samples used in the experiments
Mock biological evidence (n=8) was created by adsorbing a small quantity of blood (20µl), semen (2µl) and saliva (80µl) on flocked swabs (COPAN 4N6FLOQSwabs™ Genetics), obtaining a final mixture blood:semen:saliva of 1:10:40 on each swab.
Swabs were then air dried and prepared for sorting using the DEPArray™ Forensic SamplePrep Kit, specifically developed to simultaneously stain sperm, epithelial and white blood cells (for a detailed description of the procedure see the Instructions for Use, www.siliconbiosystems.com/en-us/Product-resources). The sample preparation procedure includes a phase where cells are detached from the support and released in solution, followed by an immunofluorescent staining to enable the detection and identification of each cell type (1).
The single cell suspension is finally loaded into the DEPArray™ Cartridge for image-based cell identification and sorting (Fig. 3 and 4).
Figure 3: Scatterplot of a DEPArray™ run: Green dots are single epithelial cells (ECs), Red dots are single sperm cells (SCs) and Yellow dots are single white blood cells (WBCs).
Figure 4: Example of single ECs (top), SCs (middle) and WBCs (bottom) images, used for the image-based cell selection at the DEPArray™.
Genotyping of single cells
In total, 52 single epithelial cells, 66 single sperm cells and 124 single white blood cells were isolated with the DEPArray™ System, for a total of 242 pure single cells.
DEPArray™-recovered single cells were resuspended in 1μl of PBS and lysed with the DEPArray™ LysePrep kit, in the same tube in which DEPArray™ recoveries have been performed.
a. Genotyping method
Multiplex PCR amplification was performed using the PowerPlex® Fusion 6C System (Promega) according to manufacturer’s instructions and standard forensic lab regulations; all the PCR runs were performed with 29 amplification cycles and including positive and negative controls, blanks and reference profiles for each of the gDNA extracted and quantified from the three different body fluids.
Capillary electrophoresis was then performed according to standard forensic procedures (Applied Biosystems 3500 Genetic Analyzer), by using GeneMapper® ID-X software for the data analysis.
b. Sensitivity and Specificity of the single cell profiling
For GeneMapper® ID-X Software, the analytic threshold for the allele call was set at 50 RFU (Relative Fluorescence Unit), the genetic analysis suitability threshold was set at allele ≥1.
Out of the total of 242 recovered single cells, 237 (98%) provided allelic amplification of at least one allele (Table 2), therefore they were considered suitable for further analysis aimed at verifying the reliability of single cells genotyping. On the other hand, 5 cells did not show allele amplification profile (2,07%) and were discarded from further analysis.
Figure 5: Example of genotyping a single epithelial cell, sperm cell and white blood cell.
Table 2: Overview of the single cells recovered (ECs: epithelial cells, SCs sperm cell WBC: white blood cells) that showed at least 1 allele call.
The total number of alleles over 50 RFU was counted for each reference gDNA (Expected Allele Count) as well as for each profile obtained from the isolated single cells (Total Allele Count). Each single cell profile was then compared to the corresponding reference gDNA profile, to assess the number of concordant alleles (Concordant Allele Count).
For each cell profile the following analysis was performed:
- Concordance %, defined as concordant alleles count / (observed allele count + allele drop-in peak count).
- Completeness % was defined as observed allele count (no drop ins included) / expected allele count.
- Aspecificity % was defined as the allele drop in peaks count + non-concordant allele count/ total allele count.
- Stutter % was calculated considering only stutter showing a peak height >15% of the corresponding real peak and computed as the stutter count / total allele count – drop in peaks count.
For single sperm cells, the completeness percentage was computed on the haploid reference profile, whereas, for the diploid cell types (WBCs and ECs), it was computed on the full diploid reference profile.
The results (Table 3, Figure 6) show that the completeness of the single cells profiles ranges from 60 to 80%, despite the low input of DNA (6,6pg for diploid cells and 3,3pg for sperm cells), without affecting the profiles accuracy, showing an average concordance of 96%, and only minimal presence of non-specific peaks and stutters.
Table 3: Completeness, Concordance, Aspecificity data obtained from the single isolated cells.
Figure 6: Completeness/Concordance/Aspecificity/Stutter. Plot showing the Completeness, Concordance, the Aspecificity and the Stutter percentages in the isolated single cells pools per cell type.
c. Peak Height/Heterozygous Peak Height Ratio Analysis
For diploid cells only, the mean of peak heights was calculated for each profile and the minimum and maximum peak height registered: from here the peak height ratios were computed for the observed heterozygous loci only and expressed in percentage. For each single cell, the mean of heterozygous peaks was calculated as the mean of the peak’s ratio percentage. For each of these parameters the overall means were calculated per cell type (EC and WBC).
The threshold to determine unbalanced allele was set at 60% following international guideline recommendations: this means that the height of the smaller peak should not be below the 60% of the height of the taller peak, in the same locus, for the same profile. For this analysis only, concordant profile alleles were considered in the elaboration, thus stutter and drop-ins were excluded, as well as AMXY and YSTRs loci.
Data analysis (Table 4, Figure 7) shows that the overall mean ratio of peak height was above the set threshold in the 76% of the heterozygous loci, confirming that the DNA obtained from single cells not only provides an almost complete profile, but also generates profiles with balanced alleles.
Table 4: Peak height and heterozygosity ration on the isolated diploid single cells
Figure 7: Heterozygous peak ratio (HPR) Plot showing the Completeness, Concordance, the Aspecificity and the Stutter percentages in the isolated single cells pools per cell type.
Summary
Single cell forensics represents the ultimate solution to the problem of mixed forensic evidence. Here we show that by combining the precision of DEPArray™ digital sorting with the accuracy and sensitivity of PowerPlex® Fusion 6C System, it is possible to generate meaningful genetic profiles, even from single cells.
Indeed, a single pure cell constitutes a perfectly complete system in which the entire genetic information is present. Even in a single cell, the DNA quantity represents the extreme condition of low template DNA: still, the genomic representation that it was possible to obtain is utterly complete, as it is unaffected by the stochastic sampling which would be unavoidable when starting from an equivalent quantity of cell-free DNA molecules.
Francesca Fontana PR&D Biology Manager, Menarini Silicon Biosystems, Italyt
Francesca Fontana focuses her activities on the development of methods for the isolation of rare cells from a variety of biological samples, as well as the genetic analysis at a single cell level. In this context, she transferred the advantage of single cells analysis to forensic genetics, for a pioneer approach to the resolution of the forensic mixtures.
References
- Fontana F et al, FSI Genetics 2017. Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach
- Meloni et al, FSI Genetics, 2019. Optimization of STR amplification down to single cell after DEPArray™ isolation
- Anslinger et al, FSI Genetics 2019. New strategies in the field of mixture deconvolution single cell STR profiling
- Schulte et al, J Forensic Sci 2023. A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology
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