Evaluation of Library Preparation Workflows and Applications to Different Sample Types Using the PowerSeq® 46GY System with Massively Parallel Sequencing.

This project evaluated the prototype PowerSeq® 46GY System using donor DNA and casework-type samples. The goal of this study was to determine whether modifications to the manufacturer's protocol could increase read coverage and improve sample results. Buccal and casework-type libraries were prepared using the TruSeq® DNA PCR-Free HT kit or the KAPA HyperPrep kit. Both kits were evaluated unmodified, and by substituting AMPure® XP beads for the beads of the most optimal kit. Two qPCR kits, the PowerSeq® Quant MS System and KAPA Library Quantification Kit, were also evaluated along with a KAPA size-adjustment workbook, which was compared as a third quantification method. Libraries were sequenced using the MiSeq® FGx and data were analyzed with STRait Razor. Results suggested that all three quantification methods overestimated library concentration, but the PowerSeq kit was most accurate. Samples prepared with the TruSeq library kit provided the highest coverage and the fewest instances of dropout and below-threshold alleles compared with the KAPA kit. Additionally, all bone and hair samples demonstrated full profile completeness, with bone samples yielding a higher average coverage than hair samples. Overall, our study demonstrated that the 46GY manufacturer's protocol produced the best quality results compared to alternative library preparation options.

Assessment of detection limits for dyed and mounted textile fibers using Raman spectroscopy.

Trace evidence in the form of textile fibers can be used to link objects and places during an investigation. Raman spectroscopy is a well-established technique that has been used for the examination of various pigments, paints, inks, and dyes. The objective of this study was to determine the capability of Raman spectroscopy to detect several different dye classes and colors on a variety of textile fibers. To test this, four categories of dyes, reactive, disperse, acid, and direct were examined with Raman microscopy while applied to one of five fiber types (cotton, polyester, nylon, wool, and rayon). Each dye category was tested using four colors, black, blue, red, and yellow, while at four concentrations of dye (w/w), 4% (black only), 1%, 0.5%, and 0.05% (blue, red, and yellow). Finally, each dye, fiber, color, and dye concentration combination were examined with Raman using one of two laser excitation sources (532 nm and 780 nm) while mounted in one of two mounting media, Permount™ and Entellan® new, as well as unmounted. Raman spectroscopy could detect some dyes at low concentrations (0.5% and 0.05%) even when mounted in mounting media and covered with a glass coverslip. Excitation source, dye category, dye concentration, fiber type, and mounting method all influence the ability to detect any given dye. These results support the continued study of Raman as a tool for the examination of fiber dyes as it has shown the potential to be effective even under constraints experienced by forensic examiners.

Combining multiplex PCR and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals.

The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.