Evaluation of the Ion AmpliSeq™ PhenoTrivium Panel: MPS-Based Assay for Ancestry and Phenotype Predictions Challenged by Casework Samples

Abstract

As the field of forensic DNA analysis has started to transition from genetics to genomics, new methods to aid in crime scene investigations have arisen. The development of informative single nucleotide polymorphism (SNP) markers has led the forensic community to question if DNA can be a reliable “eye-witness” and whether the data it provides can shed light on unknown perpetrators. We have developed an assay called the Ion AmpliSeq™ PhenoTrivium Panel, which combines three groups of markers: 41 phenotype- and 163 ancestry-informative autosomal SNPs together with 120 lineage-specific Y-SNPs. Here, we report the results of testing the assay’s sensitivity and the predictions obtained for known reference samples. Moreover, we present the outcome of a blind study performed on real casework samples in order to understand the value and reliability of the information that would be provided to police investigators. Furthermore, we evaluated the accuracy of admixture prediction in Converge™ Software. The results show the panel to be a robust and sensitive assay which can be used to analyze casework samples. We conclude that the combination of the obtained predictions of phenotype, biogeographical ancestry, and male lineage can serve as a potential lead in challenging police investigations such as cold cases or cases with no suspect.

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

“…Marker coverage across the 200 autosomal markers included in the panel ranged between 309,357 and 736,887 total reads for 20 cells, 216,401–549,980 reads for 10 cells, and 106,865–367,782 reads for 5 cells. The obtained values were comparable with the ones observed in the sensitivity study, where the total number of reads across the triplicates was 340,461–698,760, 374,692–637,041, and 94,120–395,664 for 125, 62, and 31 pg respectively [ 24 ]. The coverage for each marker is presented in Supplementary Materials, Table S1 .…”

“…The cell groups were collected to evaluate the performance of the workflow combining single-cell separation using the DEPArray™ N×T System with Next Generation Sequencing using the Ion S5. The results obtained in this study were compared to the data from a previously performed validation study [ 24 ] of the PhenoTrivium assay. The collected cell groups approximately corresponded to 100, 50, and 25 pg DNA, which were similar to the DNA input used in the sensitivity study (125, 62, and 31 pg).…”

“…Panels containing SNPs associated with phenotype [ 22 , 46 , 47 , 48 ] and ancestry [ 21 , 49 , 50 , 51 ] have been introduced, and recently assays combining those have been published [ 23 , 24 , 52 ]. Forensic DNA phenotyping is now legislated in different European countries [ 20 ], and it is expected that police investigators will seek expert opinions on samples of interest.…”

“…For all samples used in the study, genomic DNA (gDNA) was extracted with the DEPArray™ LysePrep Kit (Menarini Silicon Biosystems) directly from the cell separation tubes to make sure the input corresponds with the number of collected cells. All sample extracts were subjected to manual library preparation using the Precision ID Library Kit and IonCode™ barcode adapters using the validated Ion AmpliSeq™ PhenoTrivium Panel [ 24 ]. The amplification of the targets was carried out in the same tubes as the extraction to avoid changing the DNA input.…”

“…The HIrisPlex-S SNP set contains an indel SNP (rs796296176) in the form of an A insertion that was manually reviewed and called using IGV 2.7. Predictions were interpreted according to the PhenoTrivium validation paper [ 24 ]. For ancestry prediction, called genotypes were merged into a consensus single SNP profile using Converge, and the prediction was performed with the bootstrapping admixture analysis [ 27 ] feature of Converge using the 75% resampling size, 1000 replications, and the Precision ID Ancestry Panel Ancestry Frequency File v1.1.…”