PowerPlex Fusion: An Expanded STR Multiplex for New Global Standards. 2012, Promega Corporation.

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1 PowerPlex Fusion: An Expanded STR Multiplex for New Global Standards Kathryn Oostdik Senior Research Scientist December 12, 2012

2 Expansion of Databases Expansion of Multiplexes Recognized need for common loci Expanded European Standard Set CODIS expansion Goals More data sharing Fewer adventitious matches

3 What is the PowerPlex Fusion System? Analyzes 24 loci in a single reaction CODIS and European Standard Set (ESS) loci Penta D, Penta E, DYS391, and amelogenin One system with protocols for extracted DNA and direct amplification One kit to validate, purchase and QC Rapid thermal cycling Compatible with the Applied Biosystems 31xx and 3500 Genetic Analyzers Proven 5-dye chemistry requires no instrument/software upgrades

4 Protocols Extracted DNA, FTA punches, NonFTA punches, Swabs Extracted DNA FTA punches NonFTA Punches Swabs Extract DNA from sample Punch FTA into plate Punch card into plate, treat with PunchSolution SwabSolution incubation of swab Add up to 15µl of sample into reaction Add reaction mix Add reaction mix to dried punch Add 2µl of extract into reaction mix Cycling time ~90 minutes for all applications

5 PowerPlex Fusion Positive Control: 500pg 2800M genomic DNA

6 PowerPlex Fusion Negative Control

7 PowerPlex Fusion Allelic Ladder

8 PowerPlex Fusion Developmental Validation Validators Validators Kathryn Oostdik, Kristy Lenz Jeff Nye, Kristin Schelling Donald Yet, Scott Bruski, Joshua Strong Clint Buchanan, Joel Sutton Jessica Linner, Nicole Frazier Hays Young Learden Matthies Jeff Hahn Regina Wells Natasha Williams, Jean Johnston, Monica Price Jody Koehler Melisa Staples, Katie L. Swango Carolyn Hill Site Promega Corporation Michigan State Police Michigan State Police U.S. Army Criminal Investigation Laboratory U.S. Army Criminal Investigation Laboratory Arkansas State Police Los Angeles County Sheriffs Department Kansas Bureau of Investigation Kentucky State Police Washington State Patrol Crime Laboratory Division Texas Department of Public Safety New Hampshire State Police National Institute of Standards and Technology

9 Developmental Validation Tests Species Specificity Sensitivity Inhibitors Stability Reproducibility Case-type Samples Population Study Mixtures Precision and Accuracy Stutter Concordance Reduced Reaction Volume Cycle Number Annealing Temperature Magnesium Titration DNA Polymerase Titration Primer Titration Final Extension

10 Species Cross Reactivity Study Species Qty Cross- Reactivity Chimpanzee (male) 0.5ng Y Macaque (male) 0.5ng Y Gorilla 0.5ng Y Chicken 10ng Y Pig 10ng Y Mouse 10ng Y Cow 10ng N Cat 10ng N Dog 10ng N Rabbit 10ng N Deer 10ng N Horse 10ng N Species Qty Cross- Reactivity Escherichia coli 10ng N Enterococcus faecalis 10ng N Lactobacillus acidophilis 10ng N Streptococcus mutans 10ng N Staphylococcus epidermidis 10ng N Micrococcus luteus 10ng N Fusobacterium nucleatum 10ng N Streptococcus salivarius 10ng N Streptococcus mitis 10ng N Acinetobacter lwoffi 10ng N Pseudomonas aeruginosa 10ng N Candida albicans 10ng N Saccharomyces cervisae 10ng N

11 Species Cross Reactivity Study Negative 10ng Chicken 10ng Mouse 10ng Pig

12 % Alleles Called Average Peak Height Ratio Sensitivity Study 100% 80% 60% 40% 20% 0% 500pg 200pg 100pg 50pg Full profiles at 100pg and greater n=30

13 Sensitivity 50pg

14 Inhibitor Tolerance Titration of Common Forensic PCR Inhibitors Tannic Acid Humic Acid Hematin ng/µl 200ng/µl 50ng/µl 200ng/µl 250µM 1mM 25ng/µl 100ng/µl 25ng/µl 100ng/µl 125µM 500µM

15 % Allele Calls Inhibitors: Hematin 100% 80% 60% 40% DNA-Site 2 DNA-Site 1 FTA-Site 1 20% 0% 0uM 750uM 1000uM 1250uM 1500uM

16 % Allele Calls Inhibitors: Humic Acid 100% 80% 60% 40% DNA - Site 1+2 FTA - Site 1 20% 0% 0ng/ul 100ng/ul 200ng/ul 300ng/ul

17 % Allele Calls Inhibitors: Tannic Acid 100% 80% 60% 40% DNA - Site 1+2 FTA - Site 1 20% 0% 0ng/ul 100ng/ul 300ng/ul 500ng/ul

18 % Allele Calls Inhibitors: EDTA 100% 80% 60% DNA - Site % FTA - Site 1 20% 0% 0mM 0.4mM 0.8mM 1.0mM

19 Case-type Samples Sample type Extraction Full Profile Blood: liquid, spotted on FTA, spotted on Whatman paper EZ1, Organic Y Cigarette butts EZ1 Y Buccal swab EZ1 Y Differential extractions EZ1 Y Touch samples PrepFiler Y Blood with soil EZ1 Y Mixtures EZ1 Y Leather EZ1 N Hair Organic Y Saliva Organic Y

20 Case-type Samples: Epithelial Fraction (Differential)

21 Case-type Samples: Sperm Fraction (Differential)

22 Case-type Samples: Door handle (PrepFiler )

23 Case-type Samples: 80:10:10 Mixture (EZ1)

24 Case-type Samples: Leather (EZ1)

25 % Unique Alleles Called Mixtures 100% 80% 60% 40% Male:Male Female:Male 20% 0% 1:1 1:2 1:5 1:9 1:19 1:0

26 Mixture: 1:9

27 Standard Deviation (bases) Precision: 3500xl AMEL D1S1656 D10S1248 Penta E D18S51 CSF1PO TH01 D21S11 D5S818 DYS391 D12S391 FGA D3S1358 D2S441 D13S317 D16S539 D2S1338 Penta D vwa D7S820 TPOX D8S1179 D19S433 D22S Bases n=48

28 Standard Deviation (bases) Precision: 3130xl AMEL D1S1656 D3S1358 D2S D10S1248 Penta E D13S317 D16S D18S51 CSF1PO D2S1338 Penta D 0.06 TH01 D21S11 vwa D7S D5S818 DYS391 TPOX D8S D12S391 FGA D19S433 D22S Bases n=32

29 Stutter Minus Stutter Count Average Std Dev Mean +3 Std Dev AMEL D3S D1S D2S D10S D13S Penta E D16S D18S D2S CSF1PO Penta D TH vwa D21S D7S D5S TPOX DYS D8S D12S D19S FGA D22S Plus Stutter Count Average Std Dev Mean +3 Std Dev D22S n-2 D1S

30 Stutter ratio Stutter FL JOE TMR-ET CXR-ET Size (Bases) Stutter ratio increases with increased repeat length

31 Concordance NIST Collaboration Using proven primer sequences from PowerPlex ESI and 21 Systems Full concordance with NIST SRM No discordance with Identifiler, PowerPlex 16/21, NGM, etc. (n=652) Excluding documented null alleles in existing products

32 % Alleles Called Reaction Volume 100% 80% 60% 40% DNA 500pg DNA 100pg Swab FTA 1 pc nonfta 1pc 20% 0% 25ul 12.5ul 6.25ul DNA n=3 Swab n=4 FTA n=27 nonfta n=6

33 % Alleles Called Reaction Volume 1 and 2 Punches FTA Card 100% 80% 60% 40% Buccal 1 pc Buccal 2 pc Blood 1 pc Blood 2 pc 20% 0% 25ul 12.5ul 6.25ul 1 punch buccal n=27 1 punch blood n=3 2 punch buccal n=9 1 punch blood n=3

34 Cycle Number: Cycles FTA 26 cycles 27 cycles 28 cycles DNA: 500pg or 100pg 29, 30, 31 cycles 1 punch buccal FTA 26, 27, 28 cycles 1 punch PunchSolution -treated nonfta 25, 26, 27 cycles

35 Annealing Temperature: 57 C, 59 C, and 61 C 500pg Extracted DNA 57 C 59 C 61 C

36 Annealing Temperature 180 bases in D12S391 Extracted DNA 59 C Extracted DNA 57 C FTA 1 punch 59 C FTA 1 punch 57 C

37 Magnesium Titration Extracted DNA 500pg -20% 1X +20% Similar results with 2µl SwabSolution extract

38 Magnesium Titration FTA -20% 1 of 6 reps -20% 1X +20%

39 Magnesium Titration PunchSolution -treated nonfta -20% 1X +20% +20% 1 of 6 reps

40 DNA Polymerase Titration Extracted DNA 500pg -20% 1X +20% Similar results with FTA and nonfta punches

41 % Minus A Final Extension Extracted DNA 45% 40% 35% 30% 25% 20% 500pg 100pg n=6 15% 10% 5% 0% 0 min 10 min 20 min

42 % Minus A Final Extension Solid Support Materials 70% 60% 50% 40% 30% FTA 1 pc nonfta 1 pc Swab 20% 10% n=6 0% 0 min 10 min 20 min 40 min

43 Final Extension FTA Amelogenin, D10S min 20 min D8S min 20 min

44 PowerPlex Fusion System Developmental Validation Includes all core CODIS and ESS loci as well as Pentas and DYS391 Sensitivity: 100% alleles called at 100pg Mixture detection with ratios of 1:19 Concordant with PowerPlex ESI, 16, and 21 Reliable performance with case-type samples and against inhibitors Complete profiles detected with changes to cycle number, primer or DNA polymerase concentration Little or no increase in artifacts with lowered annealing temperature or increased magnesium Complete profiles with reduced reaction volumes

45 Resources Profiles in DNA introduction to PowerPlex Fusion article Webinar kit overview and validation update Request a free test kit, supplies limited

46 Acknowledgments Validators Validators Kathryn Oostdik, Kristy Lenz Jeff Nye, Kristin Schelling Donald Yet, Scott Bruski, Joshua Strong Clint Buchanan, Joel Sutton Jessica Linner, Nicole Frazier Hays Young Learden Matthies Jeff Hahn Regina Wells Natasha Williams, Jean Johnston, Monica Price Jody Koehler Melisa Staples, Katie L. Swango Carolyn Hill Site Promega Corporation Michigan State Police Michigan State Police U.S. Army Criminal Investigation Laboratory U.S. Army Criminal Investigation Laboratory Arkansas State Police Los Angeles County Sheriffs Department Kansas Bureau of Investigation Kentucky State Police Washington State Patrol Crime Laboratory Division Texas Department of Public Safety New Hampshire State Police National Institute of Standards and Technology

47 Acknowledgments Product Development at Promega Genetic Identity R&D Jeanne Bourdeau-Heller Marty Ensenberger Katie Oostdik Cindy Sprecher Doug Storts Project Management Susan Frackman QA Alyssa TenHarmsel Genetic Identity Production Kevin DeGrand Katie Hebble Kris Pearson Kara Raymond Ned Reimer Genetic Identity Business Unit Lotte Downey Benjamin Krenke Mary Linton Ann MacPhetridge

48 Questions

49 Supplementary Data

50 50pg 3500 Sensitivity

51 Average Peak Height Sensitivity Study (xl) 3130(xl) pg 200pg 100pg 50pg 3500(xl) n=6 3130(xl) n=21

52 Case-type Samples Blood (Organic) 500pg

53 Case-type Samples Blood (EZ1) 500pg

54 Case-type Samples Cigarette butt (EZ1) 500pg

55 Case-type Samples Rock (PrepFiler ) <500pg

56 Case-type Samples Saliva (Organic) 500pg

57 Case-type Samples Buccal Swab (EZ1) 150pg

58 Case-type Samples: Hair (Organic)

59 Primer Titration Extracted DNA 500pg -25% 1X +25%

60 Primer Titration FTA -25% 1X +25%

61 +25% Primer Concentration Extracted DNA vs FTA Extracted DNA 500pg FTA 1 punch