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Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit

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Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit, presented by Dr. Tomasz Kupiec, Head of the Forensic Genetics Section, Institute of Forensic Research, Krakow, Poland on June 14, 2018.

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Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit

  1. 1. Sample to Insight Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit Dr. Tomasz Kupiec Head of the Forensic Genetics Section Institute of Forensic Research, Kraków, Poland
  2. 2. Sample to Insight QIAGEN would like to thank our speaker, Dr. Tomasz Kupiec, for his presentation. Disclaimer: This is a QIAGEN sponsoredwebinar. QIAGEN is not affiliatedwith the Institute of Forensic Research, Kraków, Poland. The views expressedherein are those of the speaker, and do not necessarily express the views of QIAGEN.
  3. 3. 14 June 2018 Tomasz Kupiec, Andrzej Doniec, Miłosz Januła Assessment of Y chromosome degradation level using the Investigator Quantiplex Pro RGQ kit
  4. 4. - Genetic identification of human remains – introduction - Role of DNA quantification in laboratory workflow - Degradation and inhibition effects on STR analysis results - Quantification kit: sensitivity and mixture analysis - Degradation index predictive value in Y-STR and autosomal STR DNA analysis Topics discussed
  5. 5. Identification of human remains • Unidentified bodies • Disaster victims • Historical figures • Archaeological material
  6. 6. • Forensic anthropology • Fingerprints • Forensic odontology • Radiology • Medical data • Historical data • DNA testing Identification methods
  7. 7. Identification of historical figures
  8. 8. Genetic analysis of putative skull of Jan Kochanowski (1530-1584) - detection of SRY sex informative sequence – Quantifiler Duo, - amelogenin exon 2/intron 2 sex- differentiationregion (Genederplex), - NGM, PowerPlex ESI, - analysis of Y-STR and X-STR markers, - mtDNA analysis, - eyes colour prediction.
  9. 9. Victim identification workflow STAGE A: Analysis of archives STAGE B: Collecting reference STAGE C: Exhumation work Examination of Remains Examination of Evidence STAGE D: Genetic testing STAGE E: Final identification Historical data Anthropological data Victim identification workflow Source: https://www.pbgot.pl
  10. 10. Exhumation Source: https://www.pbgot.pl
  11. 11. ExhumationExhumation Source: https://www.pbgot.pl
  12. 12. Exhumation Source: https://www.pbgot.pl
  13. 13. Sample selection • The hardest part of skeleton • Positive DNA analysis results: 90-100% 80-90% 70-80% <70%
  14. 14. Laboratory Workflow Magnetic extraction Organic extraction (bones) Quantification DNA Amplification Electrophoresis X2 NGS Source:https://www.thermofisher.com Source:https://www.qiagen.com Source:https://www.qiagen.com Source:https://www.qiagen.com
  15. 15. • Autosomal markers Y Chromosome mtDNA Genetic markers inheritance
  16. 16. 16 NGS results
  17. 17. Casework sample analysis – bone samples 17 bone samples with different degradation level 0 20 40 60 80 100 1,00 1,25 1,30 1,50 2,59 3,84 3,97 4,40 5,95 7,99 9,46 10,80 15,61 18,90 22,70 27,00 125,00 %typedmarkers Degradation Index samples with DNA input below 0,5ng Amplification success rate - autosomal markers
  18. 18. Casework sample analysis – petrous bone DI=19
  19. 19. Casework sample analysis – tooth DI=8
  20. 20. Effect of humic acid (HA) - Quantifilier Trio 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 HA 0 HA 50 HA 100 HA 200 HA 400 HA 600 HA 800 HA 1000 HA neg Humic acid concentration in final volume of qPCR mixture [ng/ul] DNAquantity[ng/ul] 26 26,5 27 27,5 28 28,5 29 29,5 30 30,5 IPCCtvalue large small IPC 1 2 3 1: 1000ng/ul of HA 2: 1500ng/ul of HA 3: 2500ng/ul of HA HA concentration in PCR reaction
  21. 21. 1: 1000ng/ul of HA Inhibition
  22. 22. 2: 1500ng/ul of HA Inhibition
  23. 23. 3: 2500ng/ul of HA Inhibition
  24. 24. Investigator Quantiplex PRO RGQ • Ready to use qPCR real time assay, • Quantification: total human DNA, male DNA, • Information on degradation status: – Total human DNA, – Male DNA !!! • Information about inhibition: – new Internal Control (length 434 bp = better correlation with STR results) • Sensitivity: down to 0.1pg/ul
  25. 25. Comparison of the different qPCR quantification kits PowerQuant Investigator Quantipleks Pro RGQ Quantifiler Trio degradati on 294bp large 353bp large 214bp human 84bp small 91bp small 80bp IPC 435bp IPC 434bp IPC 130bp Y 81bp 136bp Y small 81bp Y 75bp Y large 359bp
  26. 26. Q-Rex software layout – IC analysis
  27. 27. Q-Rex software layout – autosomal DNA analysis
  28. 28. Validation experiments - Sensitivity - Stability – inhibitors: humic acid (HA) - Mixture study - Casework samples analysis – bone samples - Artificially degraded DNA samples Source:https://www.qiagen.com
  29. 29. Materials and methods – casework samples • 80 bone samples with different stage of decomposition (from 1 to 50 years after death), – DNA quantity of 13 samples was below 0,001ng/ul – 13 samples out of 67 were classified as a non-male material (female ) – 3 samples out of 54 were excluded from experiment due to quality issues
  30. 30. Materials and methods - bones preparation and extraction - PrepFiler BTA Forensic DNA Extraction Kit (Thermo Fisher Scientific) Source:https://www.thermofisher.com
  31. 31. Materials and methods - DNA quantification - Investigator Quantiplex Pro RGQ Kit (5 dye detection: green – male small, yellow – small autosomal, orange – large male, red – large autosomal, Crimson- IC) - Rotor-Gene Q Source:https://www.qiagen.com
  32. 32. Materials and methods – DNA amplification and electrophoresis Amplification: -ESSplex SE QS - 16 STRs + 2 quality markers + Amelogenin (QS1, Amelogenin, TH01, D3S1358, vWA, D21S11, QS2, D163539, D1S1656, D19S433, SE33, D10S1248, D22S1045, D12S391, D8S1179, D2S1338, D2S441, D18S51, FGA) -YFiler plus – 25 Y-STRs (DYS19, DYS385 a/b, DYS387S1 a/b, DYS389 I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635 (Y GATA C4), and Y GATA H4) Targeted DNA input: 1ng Source:https://www.thermofisher.com
  33. 33. Materials and methods – electrophoresis Electrophoresis: - ABI 3500xl ESSplex SE QS: - Analytical threshold: 200rfu (based on Qiagen recommendation) Yfiler Plus: - Analytical threshold: 334rfu (based on internal validation) Software: - GeneMapperID-X ver. 1.5 Source:https://www.thermofisher.com
  34. 34. Materials and methods – artificially degraded DNA and inhibitor resistance DNA digestion enzyme: - RNase-Free Dnase I Set (Qiagen) DNA input: - 10ng - 20ng Time of digestion: - 10’ - 20’ Inhibitor: - Humic acid (Life Technology) DNA input: - 0,1ng Inhibitor concentration: - 200 ng/ul - 400 ng/ul - 600 ng/ul - 800 ng/ul - 1000 ng/ul
  35. 35. Comparison of male and female standard quantification Name Male/Female Expected quantity [ng/ul] Measured quantity [ng/ul] Small (A) Large (A) Small (Y) Large (Y) IC [Ct] R1A* Male 5 Male NIST standard control R2A Male 0,5 0,4782 0,6466 0,4885 0,4955 15,474 R3A Male 0,05 0,0507 0,0484 0,0467 0,0457 15,978 R4A Male 0,005 0,0013 0,0012 0,0027 0,0025 15,901 R5A Male 0,0005 0,0003 0,0005 0,0002 0,0002 15,832 R1B* Female 5 Female NIST standard control R2B Female 0,5 0,3915 0,3900 Undetermined Undetermined 15,562 R3B Female 0,05 0,0431 0,0420 Undetermined Undetermined 15,657 R4B Female 0,005 0,0025 0,0028 Undetermined Undetermined 15,663 R5B Female 0,0005 0,0002 Undetermined Undetermined Undetermined 15,712 *NIST DNA standards were not quantified
  36. 36. Male/Female and Female/Male mixtures (2 repeats) Name Ratio (M:F) Expected total human DNA quantity [ng/ul] Measured quantity [ng/ul] Small (A) Large (A) Small (Y) Large (Y) IC [Ct] R1A:R1B 1:1 10 9,5196 9,8919 6,1465 6,2870 15,336 R2A:R1B 1:10 5,5 5,0522 4,3373 0,5539 0,5349 15,642 R3A:R1B 1:100 5,05 4,2500 4,1027 0,0694 0,0601 15,832 R1A:R2B 10:1 5,5 5,9531 5,8737 5,6102 5,6977 15,766 R1A:R3B 100:1 5,05 5,8618 5,6726 5,6948 5,3899 15,785 R1A:R1B 1:1 10 10,8358 10,5706 6,3617 6,7402 15,771 R2A:R1B 1:10 5,5 4,7146 4,5717 0,5639 0,5089 15,649 R3A:R1B 1:100 5,05 4,6594 3,8067 0,1001 0,0962 15,620 R1A:R2B 10:1 5,5 5,9160 6,7323 6,2766 5,7118 15,507 R1A:R3B 100:1 5,05 6,1061 7,1755 6,3999 6,1342 15,710
  37. 37. Inhibitor resistance: humic acid (HA) 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 16 17 18 19 20 21 Investigator Quantiplex Pro RGQ Kit large small IPC HA 200 HA 400 HA 600 HA 800 HA 1000 DNAconcentration [ng/ul] ICvalue[Ct] 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 16 17 18 19 20 21 Investigator Quantiplex Pro RGQ Kit large small IPC HA 200 HA 400 HA 600 HA 800 HA 1000 DNAconcentration [ng/ul] 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 16 17 18 19 20 21 Investigator Quantiplex Pro RGQ Kit large small IPC HA 200 HA 400 HA 600 HA 800 HA 1000 DNAconcentration [ng/ul]
  38. 38. autosomal [ng/ul] Y chromosome [ng/ul] repeat DNA amount [ng/ul] time of digestion [min] small small SD large large SD small small SD large large SD I 1 10 20 0,3003 0,0212 0,0102 0,0006 0,3016 0,0262 0,0069 0,00062 0,2584 0,0092 0,3034 0,0062 0,2527 0,0087 0,2470 0,00543 II 1 20 20 0,8809 0,0350 0,0484 0,0055 0,9008 0,0453 0,0225 0,00142 0,8349 0,0429 0,8943 0,0255 0,7952 0,0350 0,8016 0,02233 III 1 10 10 0,1148 0,0043 0,0036 0,0003 0,1540 0,0082 0,0040 0,00082 0,1112 0,0031 0,1618 0,0038 0,1216 0,0038 0,1739 0,00553 IV 1 20 10 0,3584 0,0068 0,0099 0,0009 0,4277 0,0234 0,0089 0,00042 0,3586 0,0103 0,3711 0,0096 0,3730 0,0120 0,4066 0,00983 DNAse I degraded samples
  39. 39. DNAse I degraded samples 0 5 10 15 20 25 30 35 40 45 50 0,0 0,2 0,4 0,6 0,8 1,0 small autosomal DNA concentration [ng/ul] autosomal DNA degradation index Y chromosome degradation index DNAconcentration [ng/ul] Degradationindex I II III IV
  40. 40. Y-STR vs autosomal STR sample
  41. 41. DI values for Y-STRs and autosomal STRs in bone samples 0 20 40 60 80 100 120 140 160 180 200 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Comparison of degradation indexes DI of autosomal DNA DI of Y chromosome DNA DI DI Y-STRs > DI autosomal STRs sample
  42. 42. Autosomal DNA DI vs STR success rate in casework samples 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0,0 10,0 20,0 30,0 40,0 50,0 60,0 70,0 80,0 90,0 100,0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Comparison of degradation indexes and identified loci (autosomal STRs) DI of autosomal DNA Investigator ESSplex SE QS DI Successrate DI value >20: loci dropouts sample
  43. 43. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0,0 20,0 40,0 60,0 80,0 100,0 120,0 140,0 160,0 180,0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Comparison of degradation indexes and analysis success rate (Y-STRs) DI of Y chromosome DNA Yfiler Plus DI Successrate Male DNA DI values vs Y-STR in casework samples DI value > 10: loci dropouts sample
  44. 44. Y-STR raw data (YFiler Plus) DI: 41,4 Number of loci: 9 out of 25
  45. 45. Y-STR (YFiler Plus)
  46. 46. Autosomal STR raw data (ESSplex SE QS ) DI: 27.4 Number of loci : 16 out of 16
  47. 47. Autosomal STR (ESSplex SE QS)
  48. 48. DI thresholds Quantifiler Trio Forensic Science International: Genetics 16 (2015) 77–85 Investigator Quantiplex PRO RGQ InvestigatorESSplex SE QS DI > 20 severely degraded YFiler Plus DI > 10 severely degraded
  49. 49. • Investigator Quantiplex Pro RGQ kit is particularly useful for the quantification of challenging samples due to its resistance to inhibition and high sensitivity, • Investigator Quantiplex Pro RGQ kit provides extensive and reliable information concerning quality of DNA sample (DI for autosomes and Y chromosome, inhibitor presence), • Male DNA degradation index and autosomal degradation index should be used separately to predict STR analysis success rate. Summary
  50. 50. The authors would like to thank Qiagen company for supplying Investigator Quantiplex Pro RGQ kit and Investigator ESSplex SE QS used in this study. Special thanks to Sylwia Chrobak from Qia-Lab for her support. Acknowledgment
  51. 51. Thank you tkupiec@ies.krakow.pl
  52. 52. Sample to Insight The Investigator Quantiplex Pro RGQ Kit is intended for molecular biology applications in forensic, human identity and paternity testing. This product is not intended for diagnosis, prevention, or treatment of a disease. Thank you for your attention! Trademarks: QIAGEN®, Sample to Insight®, Investigator®, Quantiplex® (QIAGEN Group). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. PROM-12691-001 07/2018© 2018, QIAGEN, all rights reserved. For more information, contact your QIAGEN sales representative or visit www.qiagen.com

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