Buccal swabs from all donors this website (138 males, 102 females) that had been collected over the past year were used to generate a reference database. This donor pool consisted of current employees, employee family members and former employees.

The swabs were prepared using a slight modification of the GlobalFiler Express buccal swab protocol [16]. Briefly, 300–400 μL of Prep-N-Go™ Buffer (ThermoFisher Scientific) was added to 1.5 mL Eppendorf tubes. The cotton swab was inserted into the tube with buffer and incubated at 70 °C (vs. 90 °C) in a heating block for 15 min. The lysates were used to obtain an STR profile as described below. The human male fibroblast cell line HTB-157 (ATCC, Manassas, VA), designated 1000 M, was used to prepare positive control swabs. The human embryonic palatal mesenchymal (HEPM) cell line CRL-1486™ (ATCC, Manassas, VA), designated 1000 F, was used for the mixture study. Cell culture optimization Y-27632 ic50 and scale up was performed under contract by Aragen Bioscience (Morgan Hill, CA), and cells were stored in 90% FBS, 10% DMSO at −80 °C. Cells were washed and resuspended twice in PBS buffer, quantified using a Scepter Handheld Automated Cell Counter (Millipore, Billerica, MA), and brought up to a working concentration between 200,000 and 10,000,000 cells/mL. 50 μL aliquots of the appropriate dilution of cells were added to swabs which were air dried at room temperature overnight. A

reference profile for 1000 F was obtained as described above for buccal swabs. The 1000 M cell line is the same as component Pregnenolone F in the National Institute of Standards and Technology (NIST, Gaithersburg, MD) DNA Profiling Standard SRM 2391c and the certified profile from NIST was used as the reference for concordance. Blood samples in EDTA tubes from three different donors were purchased from Memorial Blood Center (Minneapolis, MN). Two-fold serial dilutions of blood from each donor (20–2.5 μL and 1 μL) were applied to swabs. To prepare these swabs, an aliquot of each blood dilution was pipetted onto a glass slide. Then, a swab wetted with sterile water was used to recover the diluted blood from the slide. The concentration of

DNA in each blood sample was determined to calculate the amount being applied onto the swab at each dilution. DNA was extracted from 40 μL of blood from each donor using PrepFiler Forensic DNA Extraction kit (ThermoFisher Scientific) and the amount of DNA quantified in triplicate with Quantifiler Human DNA Quantification Kit (ThermoFisher Scientific) on a Applied Biosystems 7500 Real-Time PCR system v1.4 according to the manufacturer’s protocols [17] and [18]. The DNA Profiling Standard SRM 2391c, produced by NIST (Gaithersburg, MD), was used to test the accuracy of allele calls against NIST certified genotypes. For testing on the RapidHIT System, DNA from components A–D were added to the GlobalFiler Express STR reagents at 1–2 ng/20 μL.