We organized our experiment in two phases: First, we compared the two methods following the manufacturer’s protocol. Next, we redesigned the experiment starting from the same amount of bone powder for both procedures. Finally, we identified a new workflow that allows optimizing time and costs.

In the first phase, five bone samples were selected, and bone powder was extracted twice from each sample.

One set of 500mg of bone powder was washed in 10ml EDTA solution (0.5M) at room temperature and shaken. The demineralization phase takes 2-3 days, depending on bone age. After demineralization, the sample pellet was incubated in 500µl of digestion buffer G2 and pK, and DNA extracted according to the Qiagen EZ1 automatic extraction protocol¹ to obtain four eluate aliquots (E1 to E4; see Figure 2 below). Eluates can be concentrated using an Eppendorf® Concentrator-Plus (set at a temperature of 65°C) if quantification results are lower than 20pg/µl.

The second set of bone powder was processed according to the protocol for the Promega DNA IQ™ Casework Pro Kit², by adding 400µl Demineralization Buffer plus pK and thioglycerol. Only 100mg of bone powder was needed for the incubation process, which was carried out in a thermoshaker for 2.5 hours at 56°C.

Quantification of DNA extracts was performed using the Applied Biosystems® 7500 Real-Time PCR System with the Quantifiler® Trio Quantification Kit (Thermo Fisher) according to the User’s Manual³.

Amplification of the DNA extract was performed using the PowerPlex® ESX 17 System (Promega), because our previous tests⁴ showed that it provides high performance with low DNA concentrations. DNA fragment analysis was carried out on an Applied Biosystems® 3500 xL Genetic Analyzer with GeneMapper ID-X® Software v. 1.4 (Thermo Fisher).

Profiles were evaluated by comparing completeness (%) and peak mean values expressed in RFU.

In order to assess the effectiveness of the two extraction systems, we set up a second experiment that used the same starting amount of bone powder for both the systems under evaluation. Two new bone samples were identified, 800mg of bone powder was collected from each one and then divided into eight tubes: four aliquots for each protocol. In this case, it was possible to compare the quality of the two systems in terms of quantification results and peak mean values in RFU.

Starting from the data obtained, we redesigned a new workflow with the objective of achieving useful results in a single working day, instead of the 3 days required by our previous method. The new procedure starts with the preparation of four aliquots of bone powder. At first, only one aliquot of bone powder is extracted by using the Promega DNA IQ ™ Casework Pro Kit and the eluate is quantified. If the quantification result is higher than 20pg/µl, the operator proceeds with amplification and genotyping. Otherwise, if the quantification result is lower than 20pg/µl, the operator proceeds with the extraction of the remaining three aliquots of bone powder. In this way, we obtain four extracted aliquots that can be concentrated to a final volume of 20µl. The concentrated samples are finally amplified and genotyped. Figure 1 shows aschematic representation of this brand new workflow.

Figure 1. New workflow

In the first experiment, five bone samples were processed. Quantification results of the different extraction methods are depicted in Figure 2 and reported in Table 1. Note that on the X-axis of Figure 1, M1 represents Maxwell®-extracted aliquots (in purple), while E1-E4 represent EZ1-extracted aliquots (in blue). Quantification results for Sample 1 extracted using the Maxwell® System showed a concentration of 2.4pg/µl, compared to a mean of 4.3±0.9pg/µl efor aliquots extracted using EZ1. Samples 2, 3 and 4 extracted with the Maxwell® System showed a slightly better performance compared with aliquots extracted by EZ1: Sample 2 had a concentration of 22.4 pg/µl versus a mean of 8.7 ± 5.2 pg/µl with EZ1-extracted aliquots; Sample 3 concentration was 13.2pg/µl versus a mean of 8.0 ± 3.6 pg/µl; and Sample 4 concentration was 5.4 pg/µl versus a mean of 2.3 ± 1.5 pg/µl. No results were obtained from Sample 5, following either of the methods.

The experimental results, in terms of yield during the quantification and typing phase, show a comparable quality for the two extraction systems.

In order to achieve an ultimate assessment about the performance of the two systems, it is also necessary to investigate costs in terms of processing time and materials. The use of the Promega kits significantly reduces the incubation and decalcification times, saving the operator at least one working day. In addition, the reagent volumes required by the Promega kit and the smllaer number of steps to which each sample is subjected, enable a reduced risk of contamination.

On the other hand, applying our standard protocol, the starting sample is large enough (500mg of bone powder) to allow obtaining four aliquots of extract. In conditions of low concentrations (<20 pg/µl), these aliquots can be combined and concentrated to get a single concentrated extract that can be successfully amplified.

Starting from the Promega DNA IQ ™ Casework Pro Kit, the new workflow that we developed achieves useful results in just a single working day, instead of the 3 days required by our previous method.

In addition, 400mg of bone powder (four aliquots of 100 mg) is a reasonable quantity to collect, in terms of time and difficulties of the sampling process. The strategy of extracting a single 100mg aliquot allows the operator to save time and reagents if the concentration of the extract is greater than 20pg/µl. Only when the first aliquot extracted has a concentration of less than 20pg/µl, do the remaining three aliquots need to be be extracted, combined and then concentrated to obtain enough DNA for amplification to obtain a good genetic profile.

The aim of our project was to identify the best strategy to conduct genetic analysis on skeletal remains in terms of quality of results, costs and processing time. Experimental results show that the two protocols we evaluated are equivalent in terms of DNA yield and quality of the genetic profiles. Although our study utilized a limited set of data, the Bone DNA Extraction Kit and the DNA IQ™ Casework Pro Kit for the Maxwell® (16) Instrument enabled the design of a new operational workflow that simultaneously: 1) optimizes sample processing time; 2) reduces costs in terms of materials and hands-on work; 3) reduces the amount of bone powder needed for the analysis;and,4) above all, reduces the risk of contamination.

¹Qiagen EZ1® Advanced XL User Manual.

²DNA IQ™ Casework Pro kit for Maxwell®- Promega Corporation

³Quantifiler® Trio quantification kit -Thermo Fisher, User’s Manual

⁴Meloni V. (2019) Optimization of STR amplification down to single cell after DEPArrayTM Isolation. Forensic Science International: Genetics Supplement Series.