The optimisation of DNA recovery and analysis from challenging bone samples

Abstract

Two of the most important problems facing the DNA-based identification in forensic cases are the DNA degradation and the PCR inhibition. In a lot of cases, degradation and inhibition cannot be distinguished from one another when performing a short tandem repeat (STR) genotyping. This can result in repeating the DNA extraction process or considering the sample not suitable for forensic genetic analysis.
One of the most problematic forensic samples are bone samples, especially mass graves cases. Bone samples usually require grinding into fine powder and demineralisation prior to the lysis and DNA extraction process. The DNA extracted from bone samples can be degraded, contain PCR inhibitors or both.
The current study had two phases: The first phase was the pig bones (femur and rib bones) experiment (200 mg of bone powder). Conventional demineralisation (incubation of bone powder with EDTA), total demineralisation (incubation of bone powder with EDTA, proteinase k and N-Lauryl sarcosine sodium) and non-demineralised (powder only samples) were prepared, prior to the extraction processes. Six DNA extraction and precipitation methods were tested: PrepFiler® BTA Forensic DNA Kit, DNeasy® Blood and Tissue Kit, DNA IQ™ System and ChargeSwitch® Forensic DNA extraction kit along with an ethanol and isopropanol precipitation. The different extraction methods were compared based on the quantity of the extracted DNA using Qubit™ 3.0 Fluorometer and real-time PCR and the removal of PCR inhibitors using an in-house developed multiplex system.
The in-house multiplex system was able to determine the efficiency of the extraction methods and able to differentiate between DNA degradation and PCR inhibition in the DNA precipitation method. The results showed that the femur bone samples are a more suitable sample for forensic DNA analysis than rib bone samples. The results also showed that the total demineralisation technique followed by DNA extraction using the PrepFiler® BTA Forensic DNA Kit was the most efficient extraction method. The alcohol precipitation (total demineralisation) showed high concentrations of extracted DNA, but the internal amplification controls component of the multiplex system showed varying degrees of PCR inhibition.
The second phase of this study was the human bones experiment. In this experiment, human bone samples (200 samples) including femur, humerus, tibia and ulna were collected from a mass grave in the south of Iraq. The bone powder (500 mg) was subjected to total demineralisation then extracted using the PrepFiler® BTA Forensic DNA Kit. The extracted DNA was quantified using two real-time PCR kits: the Quantifiler® Trio DNA Quantification Kit and the Investigator Quantiplex Pro Kit. The DNA was then amplified using three STR kits: the PowerPlex® 21 System, the PowerPlex® Fusion 6C System and the GlobalFiler® PCR Amplification Kit. The DNA samples were also amplified using the in-house multiplex system.
The quantification results showed a consistent and a similar pattern of amplification for both kits. The femur and tibia bone samples were similar in their DNA yield and individually yielded more DNA than the humerus bone samples. For the STR results, the PowerPlex® 21 System and the PowerPlex® Fusion 6C System 24 s injection produced more useful STR profiles than the 15 s injection.
For all the kits tested (the PowerPlex® 21 System, the PowerPlex® Fusion 6C System and the GlobalFiler® PCR Amplification Kit), the femur and/or tibia bone samples produced a higher number of alleles and peak height compared to the humerus bone samples.
When comparing all the kits with each other, and the injection time was kept fixed (15 s) which is what the manufacturers’ protocols recommend, the GlobalFiler® PCR Amplification Kit was the most sensitive of all the tested kits.
The in-house Multiplex results showed the pattern of allele calling of the Multiplex system can, to a certain extent reflect the pattern of allele calling in the STR kits. The coefficient of determination (R2) between the multiplex number of alleles and the STR number of alleles for the kits studied were: PowerPlex 21 (R2 = 0.506), PowerPlex Fusion 6C 15 s (R2 = 0.53), Fusion 6C 24 s (R2 = 0.572) and the GlobalFiler kit (R2 = 0.474). The R2 value was higher than the value between the number of STR alleles and the degradation index of both quantification kits except the PowerPlex® Fusion 6C system with the Quantifiler Trio kit. For the Quantifiler Trio degradation index with: PowerPlex 21 (R2 = 0.463), PowerPlex Fusion 6C 15 s (R2 = 0.571), Fusion 6C 24 s (R2 = 0.609) and the GlobalFiler kit (R2 = 0.22). While for the Quantiplex Pro degradation index with: PowerPlex 21 (R2 = 0.418), PowerPlex Fusion 6C 15 s (R2 = 0.112), Fusion 6C 24 s (R2 = 0.092) and the GlobalFiler kit (R2 = 0.183).
This suggests that the Multiplex system is comparable, and in a lot of instances more useful, than the degradation index of both quantification kits. Also, the pattern of difference in the number of alleles among the different types of bone samples for the STR kits was consistent with the Multiplex system, although the difference was not statistically significant in the case of the GlobalFiler® PCR Amplification Kit.
The experiments also showed that the IACs detected inhibition in two samples which were amplified by the PowerPlex® 21 System and the PowerPlex® Fusion 6C systems, but not by the GlobalFiler® PCR Amplification Kit, making the behaviour of the IACs consistent with the GlobalFiler® PCR Amplification Kit. The experiment also showed that this kind of inhibition can be detected by the IACs and not detected by the IPC of the quantification kits. The PowerPlex systems showed a higher resistance to inhibition than the GlobalFiler® PCR Amplification Kit, at least in the two samples with inhibition in this study. It also showed a higher resistance to inhibitors than the PowerPlex® Fusion 6C system kit.
The results of this study have identified an optimised extraction method for bone samples; and demonstrated that the quantification and the PCR systems are sensitive and the in-house multiplex is comparable to quantification systems enabling the triage of the samples prior to quantification.