Development and Validation of MPS-Based System for Human Appearance Prediction in Challenging Forensic Samples

Abstract

Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1–5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field.

From flesh to bones: Multi‐omics approaches in forensic science

“…As a result, Inkret et al [350] recommend to conduct multiple samplings (three bones per skeleton) to overcome such limitations and to obtain a better quality consensus profile. To target the same markers included in the commercial panels, recently in-house developed panels have been created, as shown by Melchionda et al [351] and by Sguazzi et al [241]; however, they still have to be tested on skeletal remains.…”

“…To target the same markers included in the commercial panels, recently in‐house developed panels have been created, as shown by Melchionda et al. [351] and by Sguazzi et al. [241]; however, they still have to be tested on skeletal remains.…”

Forensic DNA Phenotyping: a review on SNP panels, genotyping techniques, and prediction models

“…However, this implies a longer preparation, sequencing, and analysis time [271]. As a result, the current focus is on testing SNaPshot™ panels using NGS instruments [98, 99,237,238,243,320], applying single cell sequencing and NGS to analyse mixtures and touch DNA samples [136,142,241,297], and automating analysis and result interpretation to reduce analysis time [23]. This last one would allow a better handling of the samples, increase simple size, and reduce costs and time.…”

Forensic DNA Phenotyping: Genes and Genetic Variants for Eye Color Prediction

“…There are currently available a variety of genotyping assays based on MPS technologies developed to predict EVCs from DNA recovered in a crime scene, i.e., to reconstruct the appearance traits from highly decomposed remains, the biogeographic ancestry, the estimation of the age of the subject, and the age of bone remains, which certainly allow a more comprehensive approach toward the FDP applications [11][12][13]. The development of the MPS multiplex assay and the introduction of single-cell sequencing consent to enhance the EVC prediction, especially when low amounts of DNA or mixed traces were collected [14,15]. The FDP tool consists of two components: (1) a multiplex genotyping tool to analyze several DNA markers, and (2) a validated prediction model to obtain a high probability of appearance prediction.…”