Reference samples are a vital part of the forensic analysis of deoxyribonucleic acid (DNA) evidence. Efficient processing and analysis of these sample types are required for comparative analysis of an unknown electropherogram (EPG) and forensic databasing purposes (12). These reference samples can be derived from blood swabs or cheek swabs, the latter also being known as buccal cell swabs (20, 22, 32). Buccal cells, or epithelial cells of the oral cavity, are the preferred cell type for known samples as their collection is non-invasive and painless (20-21). Buccal cell collection devices typically consist of a swab (cotton or foam) and a filter paper, commonly FTA paper (1). FTA paper contains proprietary chemicals that lyse cell membranes upon contact, trapping and stabilizing DNA for downstream processing (21, 34). FTA paper also inhibits bacterial and viral growth and protects against damage from UV radiation, nucleases and oxidation (21, 34). Some of the benefits of using FTA cards include the ability to store the cards at ambient temperature for years (21, 35, 37) and to perform direct amplification of the samples thereby removing the need to utilize DNA extraction and quantitative polymerase chain reaction (qPCR) (32, 37, 39). The EasiCollectTM (EC) and EasiCollectTM + (EC+) Buccal Sample Collection Devices (General Electric (GE) Healthcare Life Sciences, Buckinghamshire, UK) have FTA sample collection cards that contain a proprietary dye that changes color from pink to white, indicating where colorless fluids, such as saliva, were likely deposited (42). This study consisted of four phases. Phase 0 determined the optimal amplification conditions, including number of polymerase chain reaction (PCR) cycles and an appropriate capillary electrophoresis (CE) injection time for high template, single source samples obtained from FTA cards using the EC and EC+ buccal cell collection devices. Samples were obtained from EC FTA cards with a Harris 1.2-mm Uni-Core Punch and amplified using the GlobalfilerTM Express PCR Amplification Kit (Thermo Fisher Scientific, Waltham, MA) using the manufacturer’s protocol with 26, 27 or 28 PCR cycles (28). Fragment separation was achieved on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA) with 5, 15 and 25 second (s) 1.2 kiloVolt (kV) injections. Samples were analyzed on GeneMapper® ID-X v1.4 (Applied Biosystems, Foster City, CA) with an analytical threshold of 150 RFU (relative fluorescence units) (31). It was determined that amplification with 26 PCR cycles was optimal for high template, single source samples from FTA cards in this laboratory. The three injection times were utilized in the remaining phases and no other parameters were changed. In Phase 1 of this study, the optimal collection method for the EC+ device from various processes was assessed using the following collection variables: 1) a dry or saliva-wet swab; 2) a circular or up-down/side-to-side motion; 3) 2, 3 or 4 motions; and 4) swabbing of one or both cheeks. This resulted in a total of 24 distinct collection processes. We found that collection techniques that involved wetting the foam head of the EC+ device provided higher peak heights, improved heterozygote balance (Hb) and minimized the rate of drop-out in EPGs. When swabbing two cheeks versus one, the median peak heights increased, indicating an increase in transfer of cellular material onto the FTA surface. The motion of swabbing - circular or up-down/side-to-side - did not have an effect on the overall quality of the EPG data. During Phase 2a, the distribution of cellular material was assessed for two collection processes that involved swabbing of two cheeks with a wet swab four times; the variation among the methods being the motion (circular or up-down/side-to-side). Two punches taken surrounding the original punch assessed during Phase 1 showed similar average peak heights (i.e. ca. 3500 RFU at a 5 s injection on the ABI 3500 Genetic Analyzer) for both collection processes. No allelic drop-out was observed with either collection technique. Phase 2b compared the EPG signal of the EC and EC+ collection devices. The EC+ collection process used for this comparison involved rubbing a wet swab across two cheeks using four circular motions as this produced no allelic drop-out and fewer samples which saturated the CE laser detector. Therefore, this method provided more data for analysis. Samples from both devices produced comparable peak heights and PHRs above 0.6 with no allelic drop-out and stutter ratios below the thresholds set by the manufacturer (28). The EC+ device was found to be robust and provided full profiles using a minimalist sample collection method. However, the probability of drop-out increased as both the number of motions and the number of cheeks decreased. Based on this study, a collection using four circular motions divided between two cheeks with a wet swab is recommended with a 5 s, 1.2 kV injection on an ABI 3500 Genetic Analyzer, since full DNA profiles were obtained with balanced heterozygote loci, expected stutter ratios, and acceptable levels of minus A artifact. Further, it was determined that this recommended collection method resulted in high-fidelity DNA signal for up to three punches. Thus, the EC+ device is reliable, easy-to-use and non-invasive for the collection of buccal cells for known reference samples. A sample obtained from the area of transfer on an FTA card from the EC+ device can produce an EPG of the quality required for the comparison of known samples to an evidentiary profile as well as for input of the genotype into a national forensic database.