Upgrading the Workflow: Enhancing Information Recovery from Forensic Evidence. The world leader in serving science

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1 Upgrading the Workflow: Enhancing Information Recovery from Forensic Evidence The world leader in serving science

2 The GlobalFiler Kit: Going Further for Casework Powered by 6-Dye Chemistry 2

3 What is the GlobalFiler Kit? Locus CODIS (Current) Europe CODIS (Proposed) GlobalFiler Kit D13S317 X X X D7S820 X X X D5S818 X X X CSF1PO X X X D1S1656 X X X D12S391 X X X D2S441 X X X D10S1248 X X X D18S51 X X X X FGA X X X X D21S11 X X X X D8S1179 X X X X VWA X X X X D16S539 X X X X The GlobalFiler Kit is our response to the worldwide expansion of forensic locus sets, recently captured in the CODIS Core Loci Working Group s recommendation to expand the CODIS Core Loci from 13 to 20 required loci and 3 highly recommended loci. 1,2 TH01 X X X X D3S1358 X X X X AMEL X X X X D2S1338 X X X X D19S433 X X X X DYS391 X X TPOX X X X D22S1045 X X X 1. D.R. Hares, Expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012), e52-e D.R. Hares, Addendum to expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012), e135. SE33 X X X 3

4 GlobalFiler Kit: Casework Design Considerations Requirements: High sensitivity for trace/low level DNA samples Good intracolor and intralocus balance for mixture samples High tolerance to PCR inhibitors Maintain high species specificity Efficient thermal cycling conditions Optimal complementation to new and existing elements in workflow 4

5 The GlobalFiler Kit: 6-Dye Configuration Only 2 loci require primer redesign All gender markers located in the same dye channel for ease of interpretation 18 Mini-STRs Combination PI*= 5.73 x GlobalFiler Kit 10 Mini-STRs PI* = 9.2 x MiniFiler Kit 8 Mini-STRs PI* = 1.05 x % of alleles in allelic ladder <400bp 5 * Preliminary calculations based on a US Caucasian Database

6 Making of a 6-Dye STR Kit Dyes were selected based on good spectral separation PET is replaced with TAZ. SID is a new dye. 6

7 Why 6 Dye? Squeezing all the loci into a 5-dye configuration would result in multiple tradeoffs: Tradeoff #1: Multiple loci in the high molecular weight size range (>400 bp), increasing drop out with degraded and challenging samples Tradeoff #2: Several loci extending almost to 500 bp, which may cause issues with sizing and resolution, i.e. alleles labeled OL or failure to resolve microvariant alleles 7

8 Why 6 Dye? Tradeoff #3: Fewer ministrs GlobalFiler Kits include 10 STRs completely under 220 bp! 8

9 Why 6 dye? Tradeoff #4: Insufficient spacing between adjacent markers; More difficult to expand marker ranges to minimize OL allele calls alleles added to 9 GlobalFiler Kit marker ladders

10 Why 6 Dye? Tradeoff #5: Many loci would require redesigned primer sequences, leading to less concordance with data generated using the original primer sequences and with the vast majority of profiles in worldwide databases. Example: Discordant SE33 Genotypes Between Different Primer Sets NGM SElect Kit (26.2, 30.2) Other Kit A (26.3, 30.2) Other Kit B (Null, 30.2) 10

11 Improving Genotype Accuracy will Reduce Manual Edits Kit Allelic Ladder Alleles Virtual Bins Degenerate Primers Identifiler Plus & Identifiler Direct Kits NGM SElect & NGM SElect Express Kits GlobalFiler & GlobalFiler Express Kits An increase in the number of alleles and virtual bins present in the allelic ladder and the addition of degenerate primers will expand the range of alleles that can be genotyped automatically => Minimize the number of OL alleles that require manual edits 11

12 Maximizing Discrimination Kit D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vwa TPOX D18S51 D5S818 FGA D10S1248 D22S1045 D2S441 D1S1656 D12S391 SE33 PI Identifiler Kit X X X X X X X X X X X X X X X 5.01 x NGM SElect Kit X X X X X X X X X X X X X X X X 2.35 x GlobalFiler Kit X X X X X X X X X X X X X X X X X X X X X *7.12 x GlobalFiler Mini s X X X X X X X X X X *9.20 x * Preliminary calculations based on a US Caucasian Database The GlobalFiler Kit: Offers a new level of discrimination Helps reduce the risk of adventitious matches Facilitate international DNA profile comparisons 12

13 The GlobalFiler Kits GlobalFiler PCR Amplification Kit GlobalFiler Express PCR Amplification Kit A high performance, 6-dyechemistry for casework samples An ultra-high throughput, 6-dyechemistry for single source samples 13

14 Optimization of GlobalFiler Kit Reaction Conditions Reagent Volumes 3.3X Master Mix 7.5 µl 10X Primer Set 2.5 µl Sample Input 15.0 µl 25.0 µl PCR Conditions 95 C/ 1 min 94 C/ 20 sec 59 C/ 90 sec 60 C/ 10 min 29 x Greater flexibility of sample input volume to facilitate analysis of low level samples Optimized thermal cycling conditions shorten amplification conditions by > 1 hr Identifiler Plus & NGM SElect Kits GlobalFiler Kit Total Time 2 hr 25 min 1 hr 20 min 14

15 Increased Sensitivity Reagent Volumes Master Mix 7.5 µl Primer Set 2.5 µl Sample Input 15.0 µl 25.0 µl The GlobalFilerKit accommodates up to 15 μl of sample input volume to facilitate improved performance for low level samples. In studies with low-concentration control DNA, conducted by five external test labs, increasing sample input from 10uL to 15uL resulted in an average of ~ 1.5 to 2 times more alleles recovered with GlobalFiler Kit. 15

16 GlobalFiler Kit Performance Example: Sensitivity [DNA] 1 ng 500 pg 250 pg 125 pg 62.5 pg pg pg GlobalFiler Kit Yind SE33 DY3 D22 D2(S4) D12 D10 D1 FGA D5 Amel D18 TPOX vwa D19 D2(S1) D16 D13 TH01 D3 CSF D7 D21 D8 Identifiler Plus FGA D5 Amel D18 TPOX vwa D19 D2 D16 D13 TH01 D3 CSF D7 D21 D8 Greater sensitivity was observed with the GlobalFiler Kit compared to the Identifiler Plus Kit Greater sensitivity observed with the GlobalFiler Kit compared to the Identifiler Plus Kit 16

17 GlobalFiler Kit Performance Example: from Mock Casework Bone Sample Limited DNA Extracts from a challenging bone sample, known to possess limited, low quality DNA, were assayed. GlobalFilerKit returned substantially more alleles than other kits tested. GlobalFiler Kit Total Alleles Recovered from Bone Sample Run GlobalFiler Kit Identifiler Plus Kit NGM SElect Kit Ave

18 GlobalFiler Kit Performance Example: Inhibitor Tolerance The GlobalFilerKit master mix buffer has been optimized to deliver enhanced inhibition tolerance compared to earlier kits 18

19 GlobalFiler Kit Performance on Casework Samples Intracolor Balance N =

20 GlobalFiler Kit Performance on Casework Samples 20 Stutter Filters % Stutter Locus GF NGM SE ID Plus CSF1PO D10S D12S D13S D16S D18S D19S D1S D1S1656 (-2nt) D21S D22S D22S1045 (+3nt) D2S D2S D3S D5S D7S D8S DYS FGA SE SE33 (-2nt) TH TPOX vwa

21 What percentage of evidence profiles do you handle on a regular basis that are mixtures of 2 or more contributors? A. <25% B % C % D % E. >80% 21

22 What percentage of evidence profiles do you handle on a regular basis that are mixtures of 2 or more contributors? 30% 25% 28% 25% 20% 15% 17% 16% 13% 10% 5% 0% <25% 25-40% % % 5. >80% N=302 (North American Forensic DNA Analysts) 22

23 Facts about the SE33 Locus The most discrimination power of any STR locus in commercial kits highest Het obs (0.9353) lowest P I value (0.0066) highest amount of alleles and genotypes observed Ideal for mixtures more alleles seen and less allele sharing Widely Used in Europe Included in the Applied Biosystems SEFiler, SEfiler Plus and NGM SElect kits for many years Becoming more widely used internationally 23 -Hill, B et al, "Population Statistics on the Proposed Expanded U.S. Core Loci", poster at the 23rd International Symposium on Human Identification (ISHI) meeting (Nashville, TN), October 16-17, 2012

24 24 SE33 Locus Ideal for Mixtures

25 Overlay of SE33 Data from 4 individuals SE33 extremely valuable for mixture interpretation In this case, we have 4 donors and 7 peaks 25

26 Why Optimize for 1ng DNA Input? (as opposed to less) Enables lower PCR cycle # protocol which helps reduce baseline noise and impact of stochastic effects (i.e. drop out and HZ peak imbalance) Enables reliable interpretable over a wider range of DNA input Enables increased minor contributor DNA input for mixtures 26

27 Examining Mixture Ratios Theoretical input of major and minor contributors in mixture samples GlobalFiler Kit using 1ng input, other kit using.5ng DNA input) Ratio GlobalFiler Major (pg) GlobalFiler Minor (pg) Other Kit Major (pg) Other Kit Minor (pg) 19: : : : : :

28 Why Optimize for 1ng DNA Input? GlobalFiler Other Kit The 4 ng sample amplified with the other kitcould not be analyzed due to saturation Average Heterozygous Peak Height (RFU) ng 0.031ng 0.062ng 0.125ng 0.25ng 0.5ng 1ng 2ng 4ng DNA Input (ng) 28

29 Rounded Threshold Comparison 500 Average RFU of Baseline Noise + 10 Standard Deviations Blue Green Yellow Red Purple Orange Threshold (RFU) GlobalFiler Other Kit GlobalFiler Other Kit GlobalFiler Other Kit GlobalFiler Other Kit Negatives 0.25 ng 0.5 ng 1 ng 29

30 GlobalFiler Kit Customer Test Site Data 30

31 GlobalFiler Kit Five Customer Test Site Study Overview CTS1 CTS2 CTS3 CTS4 CTS5 Extraction Method EZ1 QiaAmp Magnetic Beads EZ1 Organic or EZ1 Comparative Kit Identifiler Kit SGM Plus Kit Identifiler Plus Kit NGM Kit NGM Select Kit Results Higher Recovery for GlobalFiler Kit Higher Recovery for GlobalFiler Kit Higher Recovery for GlobalFiler Kit Higher Recovery for GlobalFiler Kit Higher Recovery for GlobalFiler Kit Casework sample study performed on ~ real samples (All CE Instruments used were 3500/3500xL) 31

32 External Test Site Early Evaluation GlobalFiler Kit and Identifiler Plus Kit Comparison: Sensitivity Study DNA Mixture Study 10:1 ratio mixture of control DNA 007 and 9947a was amplified and analyzed Casework sample analysis 32

33 External Test Site Early Evaluation DNA Mixture Study Identifiler Plus Kit: 2 allele dropouts detected in 3/4 replicates. 33

34 External Test Site Early Evaluation DNA Mixture Study GlobalFiler Kit: full minor profile returned in 4/4 replicates. 34

35 DNA Input = 1000 pg Customer Test Site 1 DNA Input = 1000 pg GlobalFiler Kit Full Profile (41 alleles) Identifiler Kit Partial Profile (26 alleles) Sample: Semen (Post-Coital Vag Swab hrs) Extraction: Erase and EZ1 35

36 DNA Input = 75 pg Customer Test Site 3 DNA Input = 50 pg GlobalFiler Kit Full Profile (41 alleles) ID Plus Kit Partial Profile (27 alleles) Sample: Beer Can Swab Extraction: Magnetic Beads 36

37 Over 50% more total average alleles per sample were recovered with GlobalFiler Kit versus other kits + 56% + 54% All Samples Average Alleles Recovered per Sample Partial Profiles Average Alleles Recovered per Sample GlobalFiler Other 56 GlobalFiler Other CTS1 CTS2 CTS3 CTS4 CTS5 (Identifiler ) (SGM Plus) (Identifiler Plus) (NGM ) (NGM SElect ) CTS1 CTS2 CTS3 CTS4 CTS5 (Identifiler ) (SGM Plus) (Identifiler Plus) (NGM ) (NGM SElect ) 37

38 GlobalFiler Kit Baseline Quality Read Region 300 Test Site #1 300 Test Site #2 300 Test Site #3 No Template Controls 38

39 Workflow Integration Results from the new Quantifiler Trio DNA Quantification kit are highly predictive of GlobalFiler kit performance. 39

40 40 Quantifiler Trio and HP Kits + GlobalFiler: Improved Data Recovery with low concentration samples Average alleles per replicate shown for each input volume N = 4 Test Site 1 Test Site 2 Test Site 3 Test Site 4 Test Site 5 [DNA] = 1.5 pg/ul [DNA] = 3.1 pg/ ul Sample Input Volume Increase Sample Input Volume in 10 ul 15 ul 10 ul 15 ul Recovery Increase in Recovery x x x x x x x x x x Total x x Same samples across all sites -DNA volume varied in STR reaction Additional 5uL of DNA to the reaction delivered ~ X more alleles in low concentration samples

41 Dynamic Range of Quantifiler Trio and HP Kits: Validation Sensitivity Study Avg Measured DNA Concentration (ng/ul) SWGDAM Dynamic Range Study Male DNA at Concentrations < 10 ng/ul Trio Small Auto HP Small Auto Expected DNA Concentration (ng/ul) Quantification range 5 pg/µl 100ng/uL DNA was detected for all 3 replicates from 120ng/uL- 0.8pg/uL Measured concentration = 3 pg/ul GlobalFiler Kit* = 45 pg DNA (15ul) added to the STR reaction = 82% alleles recovered 600 *For Forensic or Paternity Use Only. 41

42 Quantifiler Trio Validation Sensitivity Study and GlobalFiler Results Upfront Sample Screening 42 Expected Quantity ng/µl Measured Quantity (SA Target) ng/µl Measured Quantity (Y Target) ng/µl Measured Quantity (LA Target) ng/µl % Alleles Recovered with GlobalFiler Kit (15ul added - avg 3 reps)

43 Sensitive detection of low DNA quantity with Quantifiler Trio Kit results in a partial profile with GlobalFiler Kit GlobalFiler Kit CT QUANTIFICATION Sensitivity 0.58 pg/ul DNA Concentration in Sample (pg/ul) Trio Sample: 0.58 pg/ul DNA (15uL Input) (1,000 RFU) 43

44 Shifts in Quantifiler Trio kit IPC are predictive of inhibitory effects on GlobalFiler kit profiles. Sample: I00 pg/µl DNA ng/µl HA GlobalFiler Kit IPC CT Shift IPC CT Shift vs. Humic Acid Levels (Sample: 100 pg/ul of DNA + HA) Humic Acid Concentration (ng/ul) in Sample Trio Trio Avg PARTIAL PROFILE (10uL Input) (2,500 RFU) 44

45 Developmental Validation is Complete Experiments were performed according to the updated and revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, Dec ). Species Specificity (SWGDAM 3.2) Sensitivity (SWGDAM 3.3) Stability (SWGDAM 3.4) Reproducibility (SWGDAM 3.5) Accuracy and Precision (SWGDAM 3.5) Population Study (SWGDAM 3.7) Mixture Study 2 (SWGDAM 3.8) Guardband PCR cycling condition (SWGDAM 3.9.2) Guardband Chemistry (SWGDAM 3.9.2) All results are documented in the kit User Guides Casework kit only 4

46 Improved Efficiency for Casework Samples Sample Preparation Quantitation/ Normalization Amplification Detection Analysis * Dependent on sample type and method * Time to Result (hrs/per samples) Total Time to to Result = = ~10-58 ~6-9 hrs hrs 46 * For Research Use Only. Not for use in diagnostic procedures.

47 Development of a Next Generation Global Multiplex Go Faster! Up to 2x faster amplification time than previous generation kits GlobalFiler Kit: ~80 minutes Enables sample to answer in as little as 6 hours Go Further! 24 loci, including 10 mini-strs, increasing discrimination power by up to 9 orders of magnitude Enhanced buffer system and sensitivity for even better performance on inhibited and low-level samples Go Global! The only kit containing all markers recommended for inclusion by the CODIS Core Loci Working Group, including all markers commonly used in Europe Maximizes global database compatibility 47

48 Samples Sample type Sample info Blood on 903 S&S paper Buccal on 903 S&S paper Total number of samples Male Female 9 7 Sample age 2 months 2 months 48

49 Amplification Protocol Reaction Mix GlobalFier Kit Direct Amp No enhancer GlobalFiler Kit Direct Amp 9.6uL enhancer PowerPlex Fusion System PnG 3uL 3uL 0 Sample 1.2mm disc 1.2mm disc 1.2mm disc* Reaction mix 7.5uL 7.5uL 5uL Primer mix 2.5uL 2.5uL 5uL Enhancer 0 9.6uL**(see slide 10) TE 12uL 2.4uL 15uL 0 *Pretreat disc with punch solution: 1. Add 10µl of PunchSolution TM Reagent to each 1.2mm punch. 2. Incubate plate at 70 C for 30 minutes or until wells are dry, without covering the plate. Test cycle numbers 24 cycles 25 cycles 26 cycles Blood on 903 Buccal on

50 Injection Protocol and Data Analysis Injection: Samples were injected at 1.2kV on 3500xl with different injection time as listed below: Injection Time 1 st round Injection CE Plate CE plate 1 12 seconds 16 seconds 20 seconds 24 seconds More Injections CE plate 2 Data Analysis: Analyze threshold set at 175 RFU. Global Filter set at 0.2. Intra-color balance calculated with Y-markers excluded. 50

51 Data Summary 1 st Round of Injections Blood on 903 Card GlobalFier Kit Direct Amp No enhancer GlobalFiler Kit Direct Amp 9.6uL enhancer PowerPlex Fusion System Injection time 24sec 24sec 24sec Total number of samples Samples with Off-scale peaks Samples with Pull-up/Stutter peaks Samples with Dropout Full profile without manual editing * Buccal on 903 Card GlobalFier Kit Direct Amp No enhancer GlobalFiler Kit Direct Amp 9.6uL enhancer PowerPlex Fusion System Injection time 24sec 24sec 24sec Total number of samples Samples with Off-scale Samples with Pull-up/Stutter peaks Samples with Dropout Samples passed CGQ * * One sample failed because of OL call on Penta E in PP Fusion 51

52 Data Summary More Injections Blood on 903 Card Injection time GlobalFiler Kit Direct Amp without enhancer 12 sec 16 sec 20 sec 24 sec GlobalFiler Kit Direct Amp with 9.6uL enhancer 12 sec 16 sec 20 sec 24 sec Total number of samples Samples with Off-scale peaks Samples with Pull-up/Stutter peaks Mixture (contaminated) SQ failed Samples with Dropout Full profile without manual editing

53 Data Summary More Injections Buccal on 903 Card Injection time GlobalFiler Kit Direct Amp without enhancer 12 sec 16 sec 20 sec 24 sec GlobalFiler Kit Direct Amp with 9.6uL enhancer 12 sec 16 sec 20 sec 24 sec Total number of samples Samples with Off-scale peaks Samples with Artifact peaks SQ failed Samples with Dropout Full profile without manual editing

54 Intra Color Balance Blood on 903 paper 1st Injection 54

55 Intra Color Balance Blood on 903 paper More Injections 55

56 Intra Color Balance Buccal on 903 paper 1st Injection 56

57 Intra Color Balance Buccal on 903 paper More Injections 57

58 Average Peak Height by Marker Blood on 903 paper -1st Injection 58

59 Average Peak Height by Marker Blood on 903 paper - More Injections 59

60 Average Peak Height by Marker Buccal on 903 paper -1 st Injection 60

61 Average Peak Height by Marker Buccal on 903 paper - More Injections 61

62 Total Number of Off-Scale Peaks from All Samples 62

63 Conclusions ICB: Adding PCR Enhancer to GF direct Amplification increased the ICB of all dyes in blood-on-903 paper samples Adding PCR Enhancer increased the Green, Red and Purple ICB in buccal-on- 903 paper samples, and slightly increase the yellow ICB, while the blue ICB did not change significantly. Peak Height Adding PCR Enhancer decreased the average peak height by 14% in blood-on- 903 paper samples, and by 40% in buccal-on-903 paper samples. Decreasing injection time from 24 seconds to 20, 16 and 12 seconds did not change the average peak height significantly. 63

64 Conclusions Off-Scale Peaks Adding PCR Enhancer decrease the number of Off-Scale peaks in both blood and buccal samples. Decreasing injection time to 12 and 16 seconds could decrease the number of Off-Scale peaks in both blood and buccal samples. Genotyping Results Adding PCR Enhancer did not significantly improve the genotyping results. Decreasing injection time did not significantly improve the genotyping results. 64