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In-Field Collection of DNA from Decomposing Remains

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In-field Collection of DNA from Decomposing Remains for More Direct Analysis: Experiences with Investigator Quality Sensors. Presented by Amy Holmes, et al., Dept of Forensic Science, Sam Houston State University, on May 3, 2018 at the QIAGEN Investigator Forum, San Antonio, TX

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In-Field Collection of DNA from Decomposing Remains

  1. 1. In-field Collection of DNA from Decomposing Remains for More Direct Analysis: Experiences with Investigator Quality Sensors Amy S. Holmes, PhD*; Ryan Gutierrez, BS; Madeline Roman, BS; David Gangitano, PhD; Sheree Hughes-Stamm, PhD Department of Forensic Science Sam Houston State University Huntsville, TX USA 7th QIAGEN Investigator Forum, May 2018; San Antonio, TX USA
  2. 2. This project was partially funded by a Graduate Research Fellowship Award #2015-R2-CX-0029 (National Institute of Justice, Office of Justice Programs, U.S. Department of Justice). The opinion, findings, conclusions, or recommendations expressed in this presentation are those of the authors and do not necessarily reflect those of the National Institute of Justice. ROLE OF FUNDING
  3. 3. Please do NOT take any photographs of slides depicting human remains
  4. 4. Disaster Victim Identification (DVI) Mass fatalities Remote locations High temperatures & humidity Limited/lack of facilities Loss of electricity Inability to perform DNA analysis for weeks/month Skeletal samples Goals Fast, simple DNA collection (in field)  First 2 weeks (or prior to skeletonization) RT storage of samples Faster sample processing for STR typing 2004 SE Asian Tsunami >220,000 deceased
  5. 5. Days of Decomposition Day 1 Day 7 Day 14 April – Average Temp 15 – 25ºC
  6. 6. Day 0 Day 4 Day 6 Day 10 Day 14 QS1 QS2 Investigator 24plex QS – 100% Success
  7. 7. Sampling Approach - Swabs Skin Muscle Processed immediatelyor stored for 1 month Traditional Direct Investigator®STRGO! Lysis Buffer QIAamp® DNA Investigator kit Investigator Quantiplex® Pro Whatman® EasiCollect™ Foam NylonCotton
  8. 8. 0 20 40 60 80 100 Skin Muscle Skin Muscle Skin Muscle Cotton Foam Nylon AllelesReported(%) Fresh Tissue STR Results - Swabs Direct PCR – GO!Traditional Analysis – QS 0 20 40 60 80 100 Skin Muscle Skin Muscle Skin Muscle Cotton Foam Nylon AllelesReported(%) 0 20 40 60 80 100 Skin Muscle Skin Muscle Skin Muscle Cotton Foam NylonAllelesReported(%) Fresh Tissue Decomposed Tissue Fresh Tissue • Full profiles with fresh tissues (skin & muscle) • Cotton swabs & the foam heads from Whatman® EasiCollectTM Devices seemed to generate the most consistent results regardless of the tissue type sampled • Direct PCR of swabs less successful from decomposed remains; Nylon – PCR inhibition, dilution required 0 20 40 60 80 100 Skin Muscle Skin Muscle Skin Muscle Cotton Foam Nylon AllelesReported(%) Fresh Tissue Decomposed Tissue
  9. 9. QS Inhibition – Nylon Swabs GO! Small Amplicon 0.0487 Large Amplicon 0.0000 Degradation I n/a IPC -0.31 Allele % 100 Small Amplicon 0.0084 Large Amplicon 0.0000 Degradation I n/a IPC -13.87 Allele % 0 • Other direct STR kits? • No DNA, PCR failure, and/or complete inhibition?
  10. 10. Dilution
  11. 11. 98% Alleles 93% Alleles 29% Alleles CottonNylonFoam DECOMPOSED QS GO! QS GO! QS GO! 100% Alleles 93% Alleles 81% Alleles QS1 QS2
  12. 12. Preservation – 1 month • No significantdifference betweenskin & muscle tissue • Significantlyfewer alleleswere recovered from samples collected & stored on Nylon 4N6FLOQSwabs for 1 month 0 20 40 60 80 100 Cotton Foam Nylon Cotton Foam Nylon Skin Muscle AllelesReported(%) Decomposed Tissues Time 0 1 Month Skin Muscle
  13. 13.  QuantifilerTrio indicateslow to moderate degradation, no inhibition  QuantiplexPro indicateshigh degradation and no inhibition ST: 0.18 LT: 0.054 DI: 3.26 IPC: -0.57 98% Alleles ST: 0.24 LT: 0.011 DI: 21.6 IPC: -0.34 98% Alleles Quantifiler Trio & GlobalFiler Quantiplex Pro & 24plex QS Confirmed by QS Examples of Reworked Samples
  14. 14. 100% Alleles 24% Alleles Other direct PCR kits - Extreme degradation, inhibition? Examples of Reworked Samples Dilution GO! Partial Profile
  15. 15. 100% Alleles 69% Alleles Dilution Examples of Reworked Samples QS imbalance GO! Partial Profile
  16. 16. Sampling Approach - Biopsy Modified TENT Buffer 10mM Tris, 10mM EDTA, 1M NaCl, 2% Tween 20 (DNA leaches into solution) Stored for up to 1, 3, & 6 months PDQeX (ZyGEM) QIAquick 1:10 Dilution (no purification) Quantifiler Trio 24plex QS 24plex GO! 2μL <20 min <30 min
  17. 17. Quantification Results 0 1 2 3 4 5 6 0 40 80 120 160 200 Fresh Early Decomposition Advanced Decomposition DegradationIndex DNAConcentration(ng/µL) Small Amplicon Quantification Degradation Index Days 0-4 Days 6-8 Days 10-14
  18. 18. 0 10 20 30 40 50 60 70 80 90 100 PDQex Tissue PDQex TENT Qiaquick Tent1:10 QS Tent 1:10 Go AverageNo.Alleles(%) Fresh Early Advanced Days 0-4 Days 6-8 Days 10-14 STR Results
  19. 19. QIAquick TENT TENT 1:10 QS PDQeX TENT PDQeX Tissue FRESH TISSUES – 6 month storage TENT 1:10 GO! 100% Alleles 100% Alleles 100% Alleles 100% Alleles 100% Alleles
  20. 20. Early Decomposition (days 6 – 8) Before/onset of Bloat Differences in Decomposition
  21. 21. QIAquick TENT TENT 1:10 QS PDQeX TENT PDQeX Tissue TENT 1:10 GO! 98% Alleles 100% Alleles 89% Alleles 84% Alleles 100% Alleles DECOMPOSED – Before/Onset of Bloat QS1 QS2
  22. 22. Advanced Decomposition (days 10 - 14) Difference in Decomposition
  23. 23. QIAquick TENT TENT 1:10 QS PDQeX TENT PDQeX Tissue TENT 1:10 GO! 98% Alleles 99% Alleles 80% Alleles 82% Alleles 100% Alleles ADVANCED DECOMPOSITION
  24. 24. 87% Alleles 20% Alleles QIAquick TENT TENT 1:10 QS PDQeX TENT PDQeX Tissue TENT 1:10 GO! 60% Alleles 42% Alleles 22% Alleles ADVANCED DECOMPOSITION
  25. 25. Low/No DNA present GO! No/little benefit in re-running sample in attempt to improve profile Re-extract? Small Amplicon 0.0001 Large Amplicon 0.0000 Degradation N/A IPC 0.049
  26. 26. QS Highly Degraded GO! Small Amplicon 8.2841 Large Amplicon 0.0066 Degradation I 1255.17 IPC 0.12 Allele % 75.56 Highly degraded, No inhibition detected Re-run based on LA or another approach? 84% Alleles
  27. 27. Large Target
  28. 28. Average of Small & Large Targets
  29. 29. • All methods preserved DNA well for relatively fresh tissues (up to 4 days in this study) • Most promising methods for collecting & storing DNA at RT from decomposed remains: • Cotton or foam swabs via incision in underlying muscle tissue • Tissue biopsy in TENT buffer rapidly purified with PDQeX 2400 System • Direct PCR approaches show some potential but require optimization • Quality indicators in the Quantiplex Pro kit can increase first-pass success rates, and Quality Sensors in the Investigator 24plex STR kits can be used to confirm best route for sample rework (or not) Conclusions
  30. 30. Acknowledgements • Department of Forensic Science, Sam Houston State University • PhD Advisor: Dr. Sheree Hughes-Stamm • Graduate Students: Carrie Mayes, Kyleen Elwick, Michelle Harrel, Esiri “Ace” Tasker, Ryan Gutierrez, Maddie Roman
  31. 31. Acknowledgements • QIAGEN® for support. Special thanks to Keith Elliot, Dr. Meredith Turnbough, Bryan Davis, Angela Cacioppo, Inga Gerard • NIJ GRF Funding #NIJ 2015-R2-CX-0029 • Applied Anatomical Research Center (AARC) • Donors and families of donors
  32. 32. Amy Holmes, PhD aes049@shsu.edu
  33. 33. References • Kraemer M, Prochnow A, Bussmann M, Scherer M, Peist R, SteffenC. Developmental validationof QIAGEN Investigator24plex QS kit and Investigator24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci. FSI Genetics (2017)Mar 12;29:9-20 • Sorensen A, Berry C, Bruce D, Gahan ME, Hughes-Stamm S, McnevinD. Direct-to-PCR tissue preservationfor DNA profiling. Int J Legal Med (2016)130: 607-13. • Sorensen A, Rahman E, Canela C, Gangitano D, Hughes-Stamm S. Preservation and rapid purificationof DNA from decomposinghuman tissue samples.FSI Genetics 25 (2016) 182-90. • Holt A, Wootton SC, Mulero JJ, Brzoska PM, Langit E, Green RL. Developmental validationof the QuantifilerHP and Trio kits for human DNA quantificationinforensic samples. FSI: Genetics 21 (2016)145-57. • InvestigatorQuantiplex Pro Field Test Handbook, Sept.2016 • Ewing MM, Thompson JM, McLaren RS, Purpero VM, Thomas KJ, Dobrowski PA, DeGroot GA, Romsos EL, Storts DR. Human DNA quantification and sample quality assessment:Developmental validationof the PowerQuant system. FSI: Genetics 23 (2016)166-77. • Scherer M, Konig M, BreitbachM, Cornelius S, Bussman M, Prochnow A, Peist R. Improving sample analysis and interpretationusing QIAGEN’s latest Investigator®STR multiplex PCR assays with a novel quality sensor. FSI: Genetics Supplement Series 5 (2015) e308-9. • Brownlow RJ, Dagnall KE, Ames CE. A comparison of DNA collectionand retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extractionmethods. J Forensic Sci 57:3 (2012)713-17. • http://www.med.navy.mil/sites/nepmu2/Documents/lab/FTA-Elute-Applications-Whatman.pdf • CaputoM, Bobillo MC, Sala A, Corach D. Optimizingdirect amplificationof forensic commercial kits for STR determination.Journal of Forensic and Legal Medicine 47 (2017)17-23.
  34. 34. Small Target Internal PCR Control Inhibition Index Inhibition flaggedby each kits’ default Investigator® Quantiplex® Pro Kit 91bp 434bp Average of standards IPC CT minus sample IPC CT < -1 Quantifiler® Trio DNA Quantification Kit 80bp 130bp Sample IPC CT minus average of standards (2-5) & NTC IPC CT > 2 PowerQuant® System 84bp 435bp Sample’s IPC is compared to a standard’s CT (based on nearest quant) > 0.3 Inhibition Study Quantiplex Pro & PowerQuant have much larger internal PCR controls Each kit has different calculation for inhibition index & different flags for determining inhibited samples
  35. 35. Degradation Index - DI Large Target Small Target DI Degradation flaggedby kits’ default: Investigator® Quantiplex® Pro Kit 353bp 91bp DNA conc. of small target divided by DNA conc. of large target > 10 Quantifiler® Trio DNA Quantification Kit 214bp 80bp 1-10, >10 PowerQuant® System 294bp 84bp > 2 Comparable small target sizes, but large target varies between kits Quantiplex Pro has the longest large target  may be more predictive of degradation

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