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Direct polymerase chain reaction (PCR) is a DNA processing method in which a sample is added directly to an amplification reaction without prior purification or quantification and has been identified as a method that may improve genotyping data obtained from low-yield touch DNA samples. The goal of the project was to generate data in support of a re-evaluation of the Federal Bureau of Investigation's (FBI) Quality Assurance Standard (QAS) 9.4 and the 2018 Forensic Science Technology Working Group (TWG) operational requirements.

The project was performed in two phases. Phase I examined the direct PCR-compatible collection methods in conjunction with mock touch DNA evidence samples on a variety of substrates. Phase II examined direct PCR of touch DNA samples that were stored at room temperature for up to six months after collection and samples that were re-sampled after initial processing. Direct PCR was performed using GlobalFiler and PowerPlex Fusion 6C amplification methods that were already validated for standard casework processing.

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

Direct polymerase chain reaction (PCR) is a DNA processing method in which a sample is added directly to an amplification reaction without prior purification or quantification and has been identified as a method that may improve genotyping data obtained from low-yield touch DNA samples. The goal of the project was to generate data in support of a re-evaluation of the Federal Bureau of Investigation's (FBI) Quality Assurance Standard (QAS) 9.4 and the 2018 Forensic Science Technology Working Group (TWG) operational requirements.

The project was performed in two phases. Phase I examined the direct PCR-compatible collection methods in conjunction with mock touch DNA evidence samples on a variety of substrates. Phase II examined direct PCR of touch DNA samples that were stored at room temperature for up to six months after collection and samples that were re-sampled after initial processing. Direct PCR was performed using GlobalFiler and PowerPlex Fusion 6C amplification methods that were already validated for standard casework processing.

The technical summary for this project can be downloaded from the National Institute of Justice project page: https://nij.ojp.gov/library/publications/improving-results-touch-dna-evidence-optimized-direct-pcr-methods.

Detection of minor DNA components in biological mixtures has increased as molecular techniques have become more sensitive, and thus, mixture deconvolution has become a major concern and topic of debate in the forensic DNA community. Deconvolution of forensic samples may be improved by sequencing microhaplotype loci as they are not subject to the amplification noise artifacts and stochastic effects that impact the commonly analyzed short tandem repeat (STR) loci. By coupling a highly discriminatory microhaplotype MPS assay with probabilistic genotyping methods such NexGenID, a novel software platform optimized for mixture deconvolution and probabilistic genotyping of sequence data, or EuroForMix, a widely used open-source probabilistic genotyping software modifiable for use with microhaplotype sequence data, this effort demonstrated an end-to-end microhaplotype analysis workflow that may be efficiently implemented by practitioners.

The proposed microhaplotype panel demonstrated high discriminatory power with combined match probabilities ranging from 9.53E-52 to 4.79E-63 and the ability to infer biogeographical ancestry. The assay proved to be sensitive down to 50 pg inputs and applicable to inhibited or degraded trace samples. Application to complex DNA mixture samples demonstrates the assay's potential to exceed minor-contributor detection when compared to STR deconvolution, help solve complex cases, increase the number of samples considered suitable for comparison, and enable retesting of cold cases where a minor contributor was assumed present but was not suitable for comparison.

This study produced six csv datasets covering microhaplotype panel construction information and a variety of sample metrics for all analyzed study samples.