Improved methods to generate high-quality DNA profiles from touch DNA samples are of considerable interest to forensic DNA laboratories. Direct polymerase chain reaction (PCR) amplification, a sample processing method in which samples are added directly to amplification reactions without prior purification or quantification, has been identified as a method that may improve the generation of genotyping data from such samples; however, laboratories in the United States are required to use standard DNA processing methods to process low-level sample types. In part, this is due to FBI Quality Assurance Standard (QAS) 9.4, which requires all unknown forensic samples to undergo human-specific DNA quantification prior to amplification of short tandem repeat (STR) loci. The goal of this 3-year study was to generate data in support of a reevaluation of QAS 9.4.

In two phases, this study examined the following: direct PCR-compatible collection methods in conjunction with mock touch DNA evidence samples on a variety of substrates (Phase I), direct PCR of touch DNA samples that were stored at room temperature for up to six months after collection with the optimum methods identified in Phase I, and direct PCR of touch DNA samples that were re-sampled after initial processing. Nine collection methods and various substrates were examined in Phase I, and three time points were examined in Phase II. In both phases, two processing methods were used: standard processing samples were extracted, quantified, and amplified in accordance with the QAS; and direct PCR samples were directly amplified. All STR profiles were assessed for overall profile quality.

This project was supported by Award No. 2019-DU-BX-0009, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect those of the Department of Justice.

The following data are provided for each sample: Quantifiler Trio small autosomal DNA concentration (ng/μl), Quantifiler Trio large autosomal DNA concentration (ng/μl), DNA yield, Quantifiler Trio degradation index (DI) value, FI value, peak height (RFU) for alleles at each locus, the number of alleles obtained, the number of alleles expected, the number of original CODIS core loci obtained, random match probability (RMP), and CODIS eligibility (yes/no). Allele calls are not provided to protect the privacy of the donors.

Forensic DNA profile index (FI): A quality score between 0.05 and 10 was also assigned to each profile based on the FI developed by Hedman et al. The FI provides a quantitative quality score for each DNA profile by taking three factors into consideration: overall peak height, peak height balance within each locus in a profile, and the profile balance across all loci of a profile. A score of 10 indicates a DNA profile of the highest quality, whereas a score of 0.05 indicates a DNA profile of the lowest quality or no profile.

CODIS eligibility: Profiles with alleles at a minimum of eight of the original CODIS core loci and match rarities of at least one in ten million were considered CODIS eligible.

PHASE I

Phase I substrates: 100% cotton denim shirt, wool blend shirt (87% merino and 13% nylon), 100% merino wool shirt, 100% polyester shirt, plastic microscope slides, metal tools, vinyl shutter samples, plastic handgun grips, 9 mm brass bullets/fired cartridge casings, foam cups, miniature concrete bricks, and unfinished wood tool handles. To indicate where the items should be held and sampled, small areas were outlined on each item, excluding the fabrics and bullets. Each shirt was distributed to one donor, who wore it for a minimum of 12 h. Non-fabric substrates were distributed to three donors, who handled items for 60 s. 1 ng buccal cell spots and single fingerprints from one donor were also prepared on plastic microscope slides.

Phase I touch DNA collection methods: (1) cotton swabs (Puritan) moistened with DNA-grade sterile water (Thermo Fisher), (2) cotton swabs moistened with 0.1% Triton X-100 (MP Biomedicals), (3) dry cotton swabs, (4) microFLOQ direct swabs (Copan) moistened with water, (5) microFLOQ direct swabs moistened with 0.1% Triton X-100, (6) dry microFLOQ direct swabs, (7) Whatman non-indicating FTA paper moistened with water, (8) Whatman non-indicating FTA paper moistened with 0.1% Triton X-100, (9) dry Whatman non-indicating FTA paper.

Phase I samples were processed with two methods: (1) standard processing with DNA extraction, concentration, and quantification prior to amplification and (2) direct PCR. The samples that underwent standard processing were extracted on a VANTAGE liquid handling system (Hamilton®) with the Investigator® STAR™ Lyse&Prep Kit (Qiagen) using 200 μL lysis volumes and eluting into 50 μL TE buffer. Samples were concentrated to 17.5 μL with Microcon® DNA Fast Flow centrifugal filter units (Millipore Sigma). Quantification was performed on the concentrated samples with Quantifiler Trio in 11 μL reactions. For direct PCR, the collected samples were placed in 96-well plates for amplification. Amplification for both the extracted and direct PCR samples was performed with Thermo Fisher’s GlobalFiler™ PCR Amplification Kit in 25 μL reactions for 29 cycles on a GeneAmp™ PCR System 9700 (Thermo Fisher). Amplification reactions containing extracted samples targeted 1 ng template DNA. Extracted samples containing less than 1 ng DNA were amplified at the maximum DNA input volume. Capillary electrophoresis (CE) was performed on a 3500xL Genetic Analyzer for Human Identification (Thermo Fisher), and data were analyzed with GeneMapper ID-X® v1.5 (Thermo Fisher) using an analytical threshold (AT) of 125 RFU and a stochastic threshold (ST) of 600 RFU.

PHASE II

Phase II included three studies: a 6-month time study, a comparison of direct PCR performed with PowerPlex Fusion 6C and GlobalFiler, an evaluation of the efficacy of resampling the substrates after standard and direct PCR processing, followed by reprocessing with standard and direct PCR methods, respectively. For the 6-month time study, the collection methods used for each substrate were determined based on their performance with standard and direct PCR processing in Phase I. Collected time study samples were processed immediately (0 months) or stored at room temperature for 3 or 6 months. For the PowerPlex Fusion 6C and GlobalFiler comparison, the 0-month time study samples for each collection method were used, and an additional set of handled substrates was prepared for cotton swab & water collection if not previously included in the time study. For the resampling study, 0-month time study samples that were collected with the method identified as the best for direct PCR in Phase I were resampled immediately after completing standard and direct PCR processing and reprocessed with standard and direct PCR methods, respectively. 

Phase II substrates: 100% cotton denim shirt, 100% merino wool shirt, 100% polyester shirt, plastic microscope slides, metal tools, vinyl shutter samples, plastic handgun grips, 9 mm brass bullets/fired cartridge casings, foam cups, miniature concrete bricks, and unfinished wood tool handles. Samples were prepared as described in Phase I.

Phase II touch DNA collection methods: (1) cotton swabs (Puritan) moistened with DNA-grade sterile water (Thermo Fisher), (2) cotton swabs moistened with 0.1% Triton X-100 (MP Biomedicals), (3) dry cotton swabs, (4) microFLOQ direct swabs (Copan) moistened with water, (5) microFLOQ direct swabs moistened with 0.1% Triton X-100, (6) dry microFLOQ direct swabs. After collection, the collectors were allowed to dry at room temperature for 24 h before processing the 0-month samples. For the 3-month and 6-month samples, cotton swab punches and microFLOQ swab heads were stored at room temperature in 96-well plates covered with AirPore tape sheets (Qiagen) for 3 or 6 months.

The collected Phase II samples were processed at three time points: immediately after collection (0 months), 3 months after collection, and 6 months after collection. Samples were processed with two methods: (1) standard processing with DNA extraction, concentration, and quantification prior to amplification and (2) direct PCR. Standard processing was performed as specified in Phase I. Amplification for both the extracted and direct PCR samples was performed with Promega’s PowerPlex Fusion 6C System in 25 μL reactions for 29 cycles on a GeneAmp PCR System 9700. CE was performed on a 3500xL Genetic Analyzer for Human Identification, and data were analyzed with GeneMapper ID-X® v1.5 using an AT of 100 RFU and an ST of 500 RFU.

Phase I and Phase II datasets are provided as .xslx and .csv files.