P027 – DNA Quantification Thresholds for MPS Analysis of Highly Degraded Skeletal Remains

10:37

Wednesday August 19^th

Station 06

Duration: 12 minutes

03. Human identification

Alicia Moré Agudo

Introduction

The genetic analysis of challenging samples represents one of the main difficulties in forensic genetics, particularly in the case of highly degraded skeletal remains. In this context, remains from the Spanish Civil War, over 80 years old, are especially relevant. DNA degradation, the presence of inhibitors, sample age, and the nature of the material significantly compromise the recovery of genetic profiles.
The efficiency of the analytical workflow largely depends on quantification criteria that enable resource optimization and maximize the recovery of informative genetic profiles. This study evaluates DNA quantification parameters to establish minimum thresholds for obtaining reliable genetic profiles from highly degraded skeletal remains.

Methodology

A total of 257 DNA samples extracted from skeletal remains were analyzed. DNA was extracted using the Automated Extraction Protocol with EZ1 Advanced XL and EZ2 Connect Fx from Qiagen, quantification was performed using Real Time PCR with Quantifiler Trio (ThermoFisher), followed by MPS sequencing using MainstAY and Signature Prep kits (Qiagen). Quantification values for Small and Large targets, as well as the degradation index, were assessed and correlated with the quality of the genetic profiles obtained.

Results

Complete or near-complete profiles were obtained in samples with values ≥0.01 ng/µL for the Small target and ≥0.003 ng/µL for the Large target.
Among the 257 samples analyzed, 55 fell below the established minimum threshold: 52 yielded no genetic profile, while 3 produced partial profiles (14–24 STRs).
Of the 202 samples above the threshold, 161 generated profiles with >24 STRs, 27 produced partial profiles (<14 STRs), and 14 yielded no profile.
Based on these findings, minimum quantification thresholds for sequencing were established.

Conclusions

Pre-sequencing DNA quantification is a key tool for decision-making in the analysis of skeletal remains. The defined threshold allows for improved resource allocation and increased laboratory efficiency.
Retrospective application of these criteria would have prevented the sequencing of 55 samples with no analytical success, optimizing workflow performance.
These criteria are applicable to forensic genetics laboratories using MPS technology for the analysis of skeletal remains.

Authors

Alicia Moré Agudo (Citogen S.L.U., Spain)
Alba Hernández Maza (Citogen S.L.U., Spain)
Sandra Pérez Pérez (Citogen S.L.U., Spain)
Isabel Navarro Vera (Citogen S.L.U., Spain)
Amaya Gorostiza (Citogen S.L.U., Spain)
Marcos Merchán Valdivia (Citogen S.L.U., Spain)

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P027 – DNA Quantification Thresholds for MPS Analysis of Highly Degraded Skeletal Remains

Introduction

Methodology

Results

Conclusions

Authors

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