The challenge of predicting human pigmentation traits in degraded bone samples with the MPS-based HIrisPlex-S system

• Published in: Forensic science international : genetics Vol. 47; p. 102301
• Main Authors: Kukla-Bartoszek, Magdalena, Szargut, Maria, Pośpiech, Ewelina, Diepenbroek, Marta, Zielińska, Grażyna, Jarosz, Agata, Piniewska-Róg, Danuta, Arciszewska, Joanna, Cytacka, Sandra, Spólnicka, Magdalena, Branicki, Wojciech, Ossowski, Andrzej
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-07-2020
• Subjects: Forensic DNA Phenotyping; massively parallel sequencing; Polish Genetic Database of Totalitarianisms Victims; prediction reliability; skeletal remains; skeletal remains; prediction reliability; Forensic DNA Phenotyping; massively parallel sequencing; Polish Genetic Database of Totalitarianisms Victims
• ISSN: 1872-4973; 1878-0326
• DOI: 10.1016/j.fsigen.2020.102301

•Prediction of human pigmentation can facilitate identification of skeletal remains•Targeted MPS allows for efficient and sensitive analysis of phenotyping SNPs•Results of FDP analysis for heavily degraded samples need careful interpretation•Pros and cons of pigmentation prediction using HPS-MPS from bones are discussed
Identification of human remains is an important part of human DNA analysis studies. STR and mitochondrial DNA markers are well suited for the analysis of degraded biological samples including bone material. However, these DNA markers may be useless when reference material is not available. In these cases, predictive DNA analysis can support the process of human identification by providing investigative leads. Forensic DNA phenotyping has progressed significantly by offering new methods based on massively parallel sequencing technology, but the frequent degradation processes observed in skeletal remains can make analysis of such samples challenging.
In this study, we demonstrate the usefulness of a recently established Ion AmpliSeqTM HIrisPlex-S panel using Ion Torrent technology for analyzing bone samples that show different levels of DNA degradation. In total, 63 bone samples at post-mortem intervals up to almost 80 years were genotyped and eye, hair and skin colour predictions were performed using the HIrisPlex-S models.
Following the recommended coverage thresholds, it was possible to establish full DNA profiles comprising of 41 DNA variants for 35 samples (55.6%). For 5 samples (7.9%) no DNA profiles were generated. The remaining 23 samples (36.5%) produced partial profiles and showed a clear underperformance of 3 HIrisPlex-S SNPs - rs1545397 (OCA2), rs1470608 (OCA2) and rs10756819 (BNC2), all used for skin colour prediction only. None of the 23 samples gave complete genotypes needed for skin colour prediction was obtained, and in 7 of them (25.9%) the 3 underperformed SNPs were the cause. At the same time, the prediction of eye and hair colour using complete IrisPlex and HIrisPlex profiles could be made for these 23 samples in 20 (87.0%) and 12 cases (52.2%), respectively.
Complete HIrisPlex-S profiles were generated from as little as 49 pg of template DNA. Five samples for which the HIrisPlex-S analysis failed, consistently failed in standard STR analysis. Importantly, the 3 underperforming SNPs produced significantly lower number of reads in good quality samples. Nonetheless, the AUC loss resulting from missing data for these 3 SNPs is not considered large (≤0.004) and the prediction of pigmentation from partial profiles is also available in the current HPS tool.
The study shows that DNA degradation and the resulting loss of data are the most serious challenge to DNA phenotyping of skeletal remains. Although the newly developed HIrisPlex-S panel has been successfully validated in the current research, primer redesign for the 3 underperforming SNPs in the MPS design should be considered in the future.