Sequencing

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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting INITIAL EVALUATION OF THE EARLY ACCESS STR KIT V1 FOR THE ION TORRENT PGM Wednesday, 1st June 18:30 La Fonda Mezzanine (2nd Floor) Poster (PS‐2a.01) Kelly A Meiklejohn 1 , Sharon Wootton 2 , Joseph Chang 2 , Chien‐wei Chang 2 , Robert E Legace 2 , Narasimhan Rajagopalan 2 , James Robertson 1 1 FBI Laboratory, 2 Thermofisher Forensic laboratories traditionally utilize commercial kits for STR genotyping, which simultaneously amplify the core CODIS STR loci and tag them with fluorescent dyes for separation by capillary electrophoresis (CE). Given the known limitations of CE typing, including the sensitivity issues with degraded DNA and the ability to deconvolute complex mixtures, massively parallel sequencing (MPS) is currently being evaluated as an additional tool for STR typing. In this study, we evaluated the Early Access STR Kit v1 for the Ion Torrent PGM using both commercially available pure native DNAs (n, 5) and forensic type DNA samples (i.e. blood, saliva and sexual fluid; n, 6). To assess reproducibility, libraries were prepared in triplicate for each sample using 1 ng of DNA as input (total n, 33). Initially we examined the performance of the Early Access STR Kit v1 using the data obtained from the five commercially available pure native DNA samples based on: 1) the depth of coverage (DoC) at each locus, and 2) the allele coverage ratio (ACR) for heterozygote loci. The average DoC among the 25 STR loci included in the panel was quite varied, ranging from 1775 – 8219X. In all loci except D13S317 and D9S2157, the ACRs were above the acceptable value of 0.5. We only observed a few instances of discordance between the PGM genotypes and those obtained using the traditional CE approach, indicating that reliable STR profiles can be obtained for both pure native and forensic type DNA samples. It is likely that with further advancements and modifications of the panel and associated analysis software prior to commercial release, instances of discordance will be infrequent. Subsequent evaluations should be focused on assessing the ability of the panel to detect mixtures at a range of ratios, along with the sensitivity limits. 57
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting DEVELOPING A PROTOCOL FOR RELIABLY OBTAINING DNA BARCODE DATA FROM BIOLOGICAL FRAGMENTS ISOLATED FROM FORENSIC-LIKE SAMPLES Wednesday, 1st June 18:30 La Fonda Mezzanine (2nd Floor) Poster (PS‐2a.02) Kelly A Meiklejohn 1 , James Robertson 2 1 FBI Laboratory/Visiting Scientist Program, 2 FBI Laboratory Soil is often collected from a subject’s tire, flatbed, shoes and shovel during an investigation of a crime. Studies documenting the biological diversity in such soil samples using MPS have primarily focused on a metagenomic approach, in which the operational taxonomic units (OTUs) are identified, not the individual species present. Given that biological organisms only inhabit specific ecosystems or habitats, identifying the individual species present in a soil sample could assist with geoattribution. DNA barcoding and the associated public databases (e.g. Barcode of Life Database BOLD and GenBank) can provide a reliable molecular approach to species‐level identification. While DNA barcode data has been obtained from pristine DNA extracted from freshly obtained specimens, no reports are available on DNA extracted from environmentally obtained samples, which may have fragmented DNA. The primary goal of our research was to develop a protocol to obtain DNA barcode data from forensic‐like samples. To reach this goal we adapted previously published protocols for pristine DNA to compromised DNA, isolated from individual biological fragments found in soil samples. After completing this, we plan to develop a multiplex protocol for DNA barcoding of several biological specimens simultaneously on a MPS platform. In late fall, soil samples were collected from 11 locations in the continental US. Where possible, 10 insect and 10 plant fragments were isolated from each sample for downstream extraction and amplification of the appropriate DNA barcoding regions (total n, 213). As expected, the quantity of extracted DNA was low (on average
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Reliable solutions for focused NGS
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Sequencing

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