PowerPlex ESX 16 and ESX 17 Fast Systems PowerPlex ESI 16 and ESI 17 Fast Systems

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1 PowerPlex ESX 16 and ESX 17 Fast Systems PowerPlex ESI 16 and ESI 17 Fast Systems Bob McLaren June 27, 2013

2 PowerPlex ESX and ESI Systems Launched September 2009 to meet requirements of expanded European Standard Set of Loci (i.e., the original 7 ESS loci plus 5 new loci (D2S441, D10S1248, D22S1045, D1S1656, and D12S391) with D2S1338, D16S539, and D19S433) Two configurations ESX: New loci as small amplicons with existing ESS loci as large amplicons ESI: New loci as larger amplicons with existing ESS loci as small amplicons Both ESX and ESI configurations available with (17 plexes) and without SE33 (16 plexes) Robust performance in presence of PCR inhibitors, but not compatible with direct-amplification 3 hour 25 minute cycling time

3 PowerPlex ESX System Configuration Amel D3S1358 TH01 D21S11 D18S51 D10S1248 D1S1656 D2S1338 D16S539 D22S1045 vwa D8S1179 FGA D2S441 D12S391 D19S433 SE33 Size (bases) ESX contains 8 autosomal loci with amplicons less than 215 bases including all 5 new loci (D3S1358, TH01, vwa, D10S1248, D1S1656, D22S1045, D2S441, and D12S391) 3

4 PowerPlex ESI System Configuration Amel D3S1358 D19S433 D2S1338 D22S1045 D16S539 D18S51 D1S1656 D10S1248 D2S441 TH01 vwa D21S11 D12S391 D8S1179 FGA SE33 Size (bases) ESI contains 4 additional loci less than 215 bases that are larger than 215 bases in ESX (D19S433, D16S539, D18S51, and D8S1179) Combining ESI and ESX: 12 loci <215bp

5 Summary of Changes to Fast Systems Completely new proprietary 5X Master Mix formulation to allow for faster cycling (4 times faster than current systems) with hot-start functionality Same autosomal primer pair sequences as in current PowerPlex ESX and ESI Systems Direct amplification enabled by using 5X AmpSolution reagent

6 Cycling Program Comparison Fast Systems Standard Systems 1 cycle 96 C / 1 min 96 C / 2 min 30 or 26 cycles 96 C / 5 sec 60 C / 35 sec 72 C / 5 sec 94 C / 30 sec 59 C / 2 min 72 C / 90 sec 1 cycle 60 C / 2 min 60 C / 45 min Ramp Rate on 9700 Max 9600 Emulation Cycle Time 50 minutes* 205 Minutes *26 cycles for direct amplification - 45min TM states that user may have to validate cycle number between cycles for their particular single source direct amplification sample type 6

7 Cycling Program Comparison Extracted DNA AmpFlSTR NGM and NGM SElect PCR Amplification Kits 1 cycle 95 C / 11 min cycles Investigator ESSplex and ESSplex SE Plus Kits 1 cycle 95 C / 5 min 30 cycles Total Time 94 C / 20 sec 59 C / 3 min 1 cycle 60 C / 10 min Total Time 2.5 hours 96 C / 10 sec 61 C / 2 min 90 min Direct Amplification AmpFlSTR NGM SElect Express PCR Amplification Kit 1 cycle 95 C / 1 min cycles 94 C / 3 sec 59 C / 16 sec 65 C / 29 sec 1 cycle 60 C / 5 min Total Time 45 min PowerPlex ESX Fast and ESI Fast Systems allow cycling in minutes for both extracted DNA and direct amplification 7

8 PowerPlex ESX Fast and ESI Fast Systems Optimized for a Variety of Sample Types Extracted DNA FTA punches NonFTA Punches Swabs Extract DNA from sample Punch card into plate Punch card into plate, treat with PunchSolution Incubate swab in SwabSolution Add up to 17.5µl DNA into reaction mix Add reaction mix + AmpSolution to punches Add reaction mix + AmpSolution to dried punches Add 2µl of swab extract into reaction mix + AmpSolution Cycling time <50 minutes for all applications

9 PowerPlex ESX Fast and ESI Fast Protocol Purified DNA 1.2mm punch or SwabSolution extract Purified DNA Amp 5µl 5X Master Mix 2.5µl 10X Primer Pair Mix 17.5µl Available Sample Volume Direct Amp 5µl 5X Master Mix 2.5µl 10X Primer Pair Mix 5µl 5X AmpSolution 12.5µl Amplification Grade Water Amplification: <1 hour GeneAmp PCR System 9700 GeneAmp PCR System 2720 Veriti Thermal Cycler (0.2ml) Detection and Analysis Applied Biosystems 3130/3130xl or 3500/3500xl Genetic Analyzer (POP-4 polymer) ABI PRISM 310 Genetic Analyzer (POP-4 for ESX Fast and POP-6 for ESI Fast) GeneMapper ID or GeneMapper ID-X Softwares

10 GeneMapper ID and ID-X Files Note that sizes of alleles for autosomal are the same as in current files. However size of amelogenin is 2 bases larger For loci that are shared with PowerPlex Fusion, we have ensured that the same virtual bins are present for microvariant alleles in the bin files for the PowerPlex ESX Fast and ESI Fast systems (total of 489 allele bins for 17 plexes: 294 real alleles and 195 virtual allele bins) Total of 74 alleles for SE33 Stutter filters are based on mean plus 3X SD from population study conducted in collaboration with NIST N-2 stutter filter added for D1S1656 and SE33 in GMIDX stutter file N+4 stutter filter added for all loci in GMIDX stutter file

11 GeneMapper ID and ID-X Files Locus # Real Alleles in Total # Allele # Virtual Bins Ladder Bins AMEL D3S TH D21S D18S D10S D1S D2S D16S D22S vwa D8S FGA D2S D12S D19S SE Total

12 PowerPlex ESX 17 Fast Stutter N-4 Stutter N+4 Stutter N-2 Stutter Locus Mean SD Mean SD Mean SD AMEL NA NA NA NA NA NA D3S NA NA TH NA NA D21S NA NA D18S NA NA D10S NA NA D1S D2S NA NA D16S NA NA D22S NA NA vwa NA NA D8S NA NA FGA NA NA D2S NA NA D12S NA NA D19S NA NA SE

13 PowerPlex ESI 17 Fast Stutter N-4 Stutter N+4 Stutter N-2 Stutter Locus Mean SD Mean SD Mean SD AMEL NA NA NA NA NA NA D3S NA NA D19S NA NA D2S NA NA D22S NA NA D16S NA NA D18S NA NA D1S D10S NA NA D2S NA NA TH NA NA vwa NA NA D21S NA NA D12S NA NA D8S NA NA FGA NA NA SE

14 Standard Deviation PowerPlex ESX 17 Fast Allelic Ladder Sizing Precision: 3130xl 0.12 ESX 17 Fast Precision, 3130xl, 16 Ladders FL JOE TMR CXR Fragment Size (bases) 14

15 Standard Deviation PowerPlex ESX 17 Fast Allelic Ladder Sizing Precision: 3500xl 0.12 ESX 17 Fast Precision, 3500xl, 12 ladders FL JOE TMR CXR Fragment Size (bases) 15

16 Standard Deviation PowerPlex ESI 17 Fast Allelic Ladder Sizing Precision: 3130xl 0.12 ESI 17 Fast Precision, 3130xl, 16 Ladders FL JOE TMR CXR Fragment Size (bases) 16

17 Standard Deviation PowerPlex ESI 17 Fast Allelic Ladder Sizing Precision: 3500xl 0.12 ESI 17 Fast Precision, 3500xl, 11 Ladders FL JOE TMR CXR Fragment Size (bases)

18 Performance with Extracted DNA

19 PowerPlex ESX 17 Fast and ESI 17 Fast 500pg 2800M DNA, 3130 ESX 17 Fast ESI 17 Fast X Axes: Bases Y Axes: 4000 RFU

20 PowerPlex ESX 16 Fast and ESI 16 Fast 500pg 2800M DNA, 3130 ESX 16 Fast ESI 16 Fast X Axes: Bases Y Axes: 4000 RFU

21 PowerPlex ESX 17 Fast and ESI 17 Fast 500pg 2800M DNA, 3500xL ESX 17 Fast ESI 17 Fast X Axes: Bases Y Axes: RFU

22 PowerPlex ESX and ESI Fast Systems Comparable Sensitivity to Current Systems 22

23 PowerPlex ESX Fast Systems Comparable Sensitivity to Current Systems 23

24 PowerPlex ESI Fast Systems Comparable Sensitivity to Current Systems 24

25 Cycler Compatibility PowerPlex ESX 17 Fast, 500pg DNA, Veriti Veriti Veriti Veriti 25

26 Cycler Compatibility PowerPlex ESI 17 Fast, 500pg DNA, Veriti Veriti Veriti Veriti 26

27 Resistance to PCR Inhibitors Fast kit comparable to standard kit Fast kits comparable in robustness to standard kits with some inhibitors and more robust to others (For each inhibitor titration, N=3) 27

28 Direct Amplification

29 Flexibility by Adding AmpSolution Separately Maintain Master Mix formulation at 5X to allow maximum extracted DNA sample volume in PCR reactions Would have needed to reduce Master Mix concentration to include active ingredient in AmpSolution Reagent Higher success in reduced reaction volumes Concentration of active ingredient in AmpSolution Reagent is higher in final amplification reaction than can be attained if added to 5X Master Mix 29

30 AmpSolution Does Not Affect Amplification: PowerPlex ESX 17 Fast Cntl: 10ng 2800M in 25ul PowerPlex ESX 17 Fast for 26 cycles AS: In presence of 1X AmpSolution AS & PS: In presence of 1X AmpSolution and 10ul PunchSolution AS & SS: In presence of 1X AmpSolution and 2ul SwabSolution 30

31 Direct Amplification of Blood on FTA Punch One 1.2mm punch, 26 cycles PowerPlex ESX 17 Fast + AmpSolution Reagent PowerPlex ESI 17 Fast + AmpSolution Reagent

32 Direct Amplification of Buccal Punches PowerPlex ESI 17 Fast System, 26 cycles One 1.2mm FTA card punch + AmpSolution Reagent One 1.2mm Bode Buccal DNA Collector punch (pretreated with PunchSolution ) + AmpSolution Reagent

33 Direct Amplification of Buccal Swab PowerPlex ESI 17 Fast System, 26 cycles OmniSwab, extracted with SwabSolution. 26 Cycles, AmpSolution

34 Direct Amplification with Reduced Reaction Volumes: Blood on FTA and nonfta 1. One 1.2mm punch from blood FTA card per amplification reaction 2. One 1.2mm punch from blood nonfta card, pretreated with 10ul PunchSolution Reagent prior to adding amplification reaction 3. Same individuals blood used on both FTA and nonfta card 34

35 Reduced Reaction Volume Direct Amplification: FTA and nonfta blood with PowerPlex ESI 17 Fast Blood on FTA 12.5µl Blood on nonfta 12.5µl 25µl 25µl 35

36 Concordance

37 PowerPlex ESX 17 Fast Concordance: NIST Study There was no discordance between ESX 17 and ESX 17 Fast ESX17 Fast results compared to NIST final data set with 670 unrelated individuals (NIST U.S. population set (654 samples), SRM 2391b (10 samples) and SRM 2391c (6 samples)) ESX17 Fast Discordance with other STR Kits at: Amelogenin (1/654) SE33 (1/654) D16S539 (1/654)

38 PowerPlex ESX 17 Fast Discordance at Amelogenin ESX17 Fast ESSplex Plus (Y,Y) (X,Y) Note: NGM SElect shows same X allele dropout. This was also seen in original PowerPlex ESX 17 System concordance study performed by NIST

39 PowerPlex ESX 17 Fast Discordance at D16S539: PHR Imbalance ESX17 Fast ESI 17 Fast (12,13) (12,13) Note: Same issue seen between PowerPlex ESX 17 and ESI 17 in original concordance study (Hill et al., (2011) FSI-Genetics, vol. 5, pp )

40 PowerPlex ESX 17 Fast Discordance at SE33: Allele Dropout Relative to ESI 17 Fast ESX17 Fast ESI 17 Fast (19,19) (19,25.2) Note: Same issue seen between PowerPlex ESX 17 and ESI 17 Pro (McLaren et al., (2013) FSI-Genetics, vol. 7 pp ). Known mutation that only affects downstream primer-binding site for ESX SE33

41 PowerPlex ESI 17 Fast Concordance: NIST Study There was no discordance between ESI 17 Pro and ESI 17 Fast ESI 17 Fast results compared to NIST final data set with 672 unrelated individuals (NIST U.S. population set (656 samples), SRM 2391b (10 samples) and SRM 2391c (6 samples)) ESI 17 Fast Discordance with other STR Kits at: Amelogenin (1/656) (same as described for ESX 17 Fast) SE33 (1/656) (with ESX 17 Fast as described above) D16S539 (1/656) (with ESX 17 Fast as described above)

42 Kit Configurations 42

43 Kit Sizes and Catalog Numbers System PowerPlex ESX 17 Fast System PowerPlex ESI 17 Fast System PowerPlex ESX 17 Fast System and PowerPlex ESI 17 Fast System Bundle PowerPlex ESX 16 Fast System PowerPlex ESI 16 Fast System PowerPlex ESX 16 Fast System and PowerPlex ESI 16 Fast System Bundle Catalog # 400rxn 100rxn DC1710 DC1711 DC1720 DC1721 DC1730 DC1731 DC1610 DC1611 DC1620 DC1621 DC1630 DC

44 What s Included Pre-Amp Components System PowerPlex ESX 17 Fast PowerPlex ESI 17 Fast PowerPlex ESX 16 Fast PowerPlex ESI 16 Fast 5X Master Mix PowerPlex ESI/ESX Fast 5X Master Mix Same for all 4 Fast systems 10X Primer Pair Mix Control DNA Water PowerPlex ESX 17 Fast 10X Primer Pair Mix PowerPlex ESI 17 Fast 10X Primer Pair Mix PowerPlex ESX 16 Fast 10X Primer Pair Mix PowerPlex ESI 16 Fast 10X Primer Pair Mix New AMEL primer pair; autosomal STR primer pairs are the same as standard systems 2800M Control DNA, 10ng/μl No change Water, Amplification Grade No change 44

45 What s Included Post-Amp Components System Allelic Ladder Mix ILS PowerPlex ESX 17 Fast PowerPlex ESI 17 Fast PowerPlex ESX 16 Fast PowerPlex ESI 16 Fast PowerPlex ESX 17 Fast Allelic Ladder Mix PowerPlex ESI 17 Fast Allelic Ladder Mix PowerPlex ESX 16 Fast Allelic Ladder Mix PowerPlex ESI 16 Fast Allelic Ladder Mix New AMEL fragments; Autosomal STR fragments are the same as standard systems CC5 Internal Lane Standard 500 Pro Same for all 4 Fast systems and ESI 17 Pro Technical Manual TMD040 TMD041 TMD043 TMD044 45

46 Ancillary Products For Direct Amplification Size, rxn Catalog # SwabSolution Kit For swabs Cotton and OmniSwab 100 DC8271 PunchSolution Kit For nonfta cards - Bode Buccal DNA Collector and S&S 903 Specimen Collection paper 100 DC9271 5X AmpSolution Reagent For FTA and Indicating FTA cards 500 DM Dye Matrix Standards Size Catalog # PowerPlex 5-Dye Matrix Standards, µl (each dye) DG4600 PowerPlex 5-Dye Matrix Standards, 3100/ µl (each dye) DG

47 Features & Benefits Key Features Short amplification time Compatible with both casework and databasing samples Two formats (ESX and ESI) with different mini-str loci More small loci Largest input sample volume Large allelic ladder range STR primer pairs are the same as standard kits Meets ENFSI recommendations Benefit Quicker results More streamlined laboratory processes; simplified QC and inventory management Easily confirm results from poor-quality samples Better profiles from degraded samples Better profiles from dilute samples More accurate calls of rare alleles Concordance allows use of current databases Data can be shared across borders 47

48 Developmental Validation 1. Following SWGDAM Guidelines (Revised Dec 2012) 2. Collaboration with European labs and NIST 3. Developmental validation will be conducted on all four systems and published in one peer reviewed paper 4. Goal is to submit by end of year to peer reviewed journal

49 Summary 1. Same autosomal primer sequences as in current PowerPlex ESX and ESI standard cycling systems. No concordance issues 2. Much faster cycling (4-fold faster) than current formulations with extracted DNA at 30 cycles 3. Sensitivity comparable to current cycling systems 4. Comparable robustness to PCR inhibitors 5. Capable of direct-amplification with same time as NGM SElect 6. CE protocols for 310, 31XX and 35XX instruments

50 Acknowledgements R&D Jaynish Patel Jeanne Bourdeau-Heller Bob McLaren Jonelle Thompson Doug Storts Project Manager Susan Frackman Marketing Rohaizah James Ann MacPhetridge Lotte Downey Manufacturing QA Katie Hebble Kara Raymond Jackie Kinney Charlie Stollberg Sarah Bettinger Todd Specher Ned Reimer Kris Pearson Susan Wigdal Scientific Communications Terri Sundquist NIST Becky Hill David Duewer Margaret Kline John Butler 50

51 Questions? 51

52 Thank You for Attending Thanks to today s speaker Dr. Robert S. McLaren and Promega for their sponsorship. Dr. Robert S. McLaren Senior Research Scientist II Promega Presented by: In Association with: