Powerquant system

DNA extraction was performed using DNA IQ Casework Pro Kit and Casework Extraction Kit in the Maxwell 16® instrument according to the manufacturer’s instructions (Promega, Mannheim, Germany), resulting in an extraction volume of 50 µl. DNA concentration of samples was established by real-time PCR using the PowerQuant™ System (Promega) according to the manufacturer’s instructions providing a reproducible and reliable detection threshold at least down to 25 pg DNA [8 (link)]. Using 2 µl DNA-containing solutions, each sample was analyzed in duplicates. Bisulfite conversion was performed applying MethylEdge Conversion System Kit (Promega) corresponding to the manufacturer’s instructions with an increased elution volume of 20 µl. An initial DNA amount of 50 ng was used in the conversion. DNA amplification of age estimation CpGs [9 (link)] as well as candidate CpGs for body fluid identification [10 (link), 11 (link)] was done using PyroMark® PCR Kit (Qiagen) following the manufacturer’s instructions, adapted to an increased number of 50 cycles. One of the two PCR primers was biotinylated.
Sequence analysis was established in a PyroMark® Q48 Autoprep instrument using the PyroMark® Q48 Advanced CpG Reagent Kit according to the manufacturer’s instructions (Qiagen). Every sample and CpG site was analyzed at least twice.

DNA from blood samples was extracted using the Maxwell® RSC Blood DNA Kit on the Maxwell® 16 Forensic Instrument (Promega, Mannheim, Germany) and eluted in a final volume of 50 µL. Per sample, four spots of 40 µL blood were extracted separately and the eluates were subsequently pooled to a total volume of 200 µL. DNA from saliva samples was extracted using Maxwell® FSC DNA IQ™ Casework Kit (Promega, Mannheim, Germany). Samples were quantified using Promega’s PowerQuant® System using 5 µL of PowerQuant 2 × MM, 0.5 µL of 20 × Primer/Probe/IPC-Mix, 3.5 µL HPLC, and 2 µL of the sample. Prior to NGS library construction, DNA was sheared mechanically to allow analysis of natural strand breaks at positions weakened due to depurination in future analyses of these datasets [18 (link)]. Shearing to 250 bp was performed following the manufacturer’s guideline for the Covaris® M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA) with a duty cycle of 10%, intensity 4, and 200 cycles per burst for 80 s. After shearing, the DNA was quality checked by 2100 Bioanalyzer Instrument with High Sensitivity DNA Kit (Agilent, Waldbronn).

Samples were quantified using the PowerQuant^® System (Promega) on the 7500 RealTime PCR System (Applied Biosystems) and amplified using PowerPlex^® Fusion 6C (Promega) kit with 1.0 ng template DNA input in a final reaction volume of 25 µl. Applied Biosystem^® Veriti 96-Well Thermal Cycler (ThermoFisher) with a 29 PCR cycles program was used for the amplification. Negative and positive controls were run with the samples. Capillary electrophoresis was performed on the Applied Biosysetm^® 3500xL Genetic Analyzer (ThermoFisher) with an injection time of 24 s and a voltage of 1.2 kV.