DOI: 10.5061/dryad.mcvdnck4p
title: Improving results from touch DNA evidence with optimized direct PCR methods

description: 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.
description: 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.
 <strong>PHASE I</strong> 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.
 <strong>PHASE II</strong> 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.
description: Phase I and Phase II datasets are provided as .xslx and .csv
 files.