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Development of a Next Generation Multiplex

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  • 1. Development of a Next GenerationGlobal MultiplexHuman Identification, Life Technologies
  • 2. Outline  Evolution of Application-Specific Solutions − From the MiniFiler™ Kit to the GlobalFiler™ Kits  Next-Generation PCR Amplification: The GlobalFiler™ Kits − Designing a Global STR Multiplex Kit − Prototype Kit Development − Data Collection & Data Analysis Software Updates10/2/2012 | © Life Technologies™ 2
  • 3. Evolution of Application-Specific Solutions10/2/2012 | © Life Technologies™ 3
  • 4. Life Technologies Forensic Kit Developments: A Decade in Review Enhanced assay Streamlined Improved sensitivity & Expanded & improved One-size-fits-all for challenging single-source discrimination power for application-specific sample approach samples sample processing all casework samples processing capabilities 2001 2007 2009 2010 2012 Identifiler® Direct Identifiler® Plus Kit NGM SElect™ Identifiler® Kit MiniFiler™ Kit Kit NGM SElect™ Kit Express Kit Applied Biosystems First commercially First commercially Large multiplexes First commercially first single- available kit to available direct that incorporate available fast and amplification employ mini-STR amplification kit more robust buffer direct amplification multiplex to cover loci (<200 bp) systems, improving kit all 13 CODIS core exclusively to Eliminates performance on a loci improve extraction, wide range of GlobalFiler™ performance on purification and challenging Express Kit degraded samples quantitation steps casework samples for single source First commercially First Applied samples available 6-dye fast Biosystems kit to and direct utilize more robust amplification kit buffer system to improve results from inhibited samples10/2/2012 | © Life Technologies™ 4
  • 5. The MiniFiler™ Kit Successfully Amplifies Severely Degraded Samples  Used to conduct DNA analysis on the mummified remains in King Tutankhamen’s tomb  Enabled investigators to construct a 5-generation pedigree of the pharaohs immediate lineage  The Romanov family, the last Russian monarchy, was executed in 1918  Remains of 5 family members were officially discovered in a mass grave in 1991, but two children were missing  In 2007, the remains of two children were discovered nearby, and the MiniFiler™ Kit helped to identify them as those of the missing Romanov children10/2/2012 | © Life Technologies™ 5
  • 6. 60 Year Old Bone Sample from World War II Identifiler® Kit MiniFiler™ Kit Data courtesy of Tim McMahon, Armed Forces DNA Identification laboratory10/2/2012 | © Life Technologies™ 6
  • 7. The MiniFiler™ Kit: A Powerful Adjunct System Missing D21, D7, CSF, D16, D2, D18, FGA Identifiler® Kit D18 D21 D16 CSF MiniFiler™ Kit D2 D7 FGA10/2/2012 | © Life Technologies™ 7
  • 8. A New Approach: Application-Specific Kits The Next Generation of STR Kits High throughput chemistries High performance chemistries for single-source sample for casework applications applications Identifiler® Plus Kit NGM™ Kit Identifiler® Direct Kit NGM SElect™ Kit NGM SElect™ Express Kit10/2/2012 | © Life Technologies™ 8
  • 9. Casework Sample Workflow Improvements in Performance .10/2/2012 | © Life Technologies™ 9
  • 10. Improved Performance on Casework Samples The Identifiler® Plus, NGM™ & NGM SElect™ Kits  Robust, single amplification STR multiplex kits optimized for use with challenging casework samples  Combine the latest advances in primer synthesis, buffer optimization and thermal cycling optimization to deliver high sensitivity and improved STR performance  The NGM™ and NGM SElect™ Kits were developed specifically in accordance with new multiplex requirements stipulated by the ENDAP/ENFSI groups for use in laboratories that will utilize the expanded European Standard Set of loci (ESSL)10/2/2012 | © Life Technologies™ 10
  • 11. Improved Performance on Casework Samples The Identifiler® Plus Kit The Case:  A series of homicides in June 2010 occurred in Shangdong, China and evidence testing using the Identifiler® kit did not yield a profile  Killer breaking in through windows and attacking victims with an axe  Shangdong Province laboratory was an early adopter of the Identifiler® Plus kit and re-tested the evidence10/2/2012 | © Life Technologies™ 11
  • 12. Improved Performance on Casework Samples The Identifiler® Plus Kit The Data: Identifiler® Kit = No DNA Profile Identifiler® Plus Kit = Full Profile  Profiles generated from blood on the murder weapon, collected 10 days after the murder was committed  Four cases were connected through DNA evidence and a suspect was identified  The offender was convicted and given life in prison for his crimes10/2/2012 | © Life Technologies™ 12
  • 13. Improved Performance on Casework SamplesThe NGM™ Kit Bone sample amplified using the Bone sample amplified using the Identifiler® Kit NGM™ Kit10/2/2012 | © Life Technologies™ 13
  • 14. Single-Source Sample Workflow Improvements and Optimization .10/2/2012 | © Life Technologies™ 14
  • 15. Improved Efficiency for Single Source Samples The Identifiler® Direct & NGM SElect™ Express Kits  A new type of chemistry capable of amplifying directly from single source samples on treated paper, untreated paper and swab substrates  Removes the need for extraction/ purification and quantitation, reducing the cost and time to result associated with single source samples  Optimised to generate high quality data equivalent to that obtained from purified samples − Delivers a high first pass success rate, minimising the need for time consuming sample repeats − Facilitates processing using an optimised Expert System Software  The Identifiler® Direct kit pioneered direct amplification – the NGM SElect™ Express Kit now accelerates the process, completing amplification in ~ 45 minutes10/2/2012 | © Life Technologies™ 15
  • 16. NGM SElect™ Express Kit Optimisation Example of Good Intracolour Balance 85% 77% 59% 70%10/2/2012 | © Life Technologies™ 16
  • 17. NGM SElect™ Express Kit Optimisation Effect of Thermal Cycling Conditions: Extension Temperature 61oC 63oC 65oC 67oC Higher Temperature = D2S441 ↓ SE33 ↑10/2/2012 | © Life Technologies™ 17
  • 18. Improved Efficiency for Single Source SamplesThe NGM SElect™ Express Kit Blood on FTA® Classic Card Buccal on Copan NUCLEIC-CARD™ Buccal on Bode Buccal 4N6FLOQswabs™ Buccal on Copan10/2/2012 | © Life Technologies™ 18
  • 19. New Global Multiplex Systems: The GlobalFiler™ Kits .10/2/2012 | © Life Technologies™ 19
  • 20. What is GlobalFiler™?  The GlobalFiler™ Kits are our response to the CODIS Core Loci Working Group’s recommendation to expand the CODIS Core Loci from 13 to 21 required loci and 4 optional loci Originally communicated as a letter to the editor (FSI Genetics) and accepted for publication 4/6/11 Authored by Doug Hares from the FBI Expectation of a single amplification kit10/2/2012 | © Life Technologies™ 20
  • 21. Why a Global Multiplex? New CODIS Expanded Missing Persons Recommendations ESSL Investigations Increase international data compatibility Why? Increase discrimination power Reduce the likelihood of adventitious matches Increase the number of loci How? Incorporate loci contained in global databases10/2/2012 | © Life Technologies™ 21
  • 22. MINIMUM REQUIRED LOCI D13S317 Identifiler® Kit Recommended CODIS Core D7S820 Identifiler® Kit Loci (April 2011) D5S818 Identifiler® Kit CSF1PO Identifiler® Kit  New recommendation drops TPOX D1S1656 NGM SElect™ Kit from the list of required loci D12S391 NGM SElect™ Kit D2S441 NGM SElect™ Kit  The new list of required loci includes D10S1248 NGM SElect™ Kit all the markers from the NGM SElect™ D18S51 Identifiler® & NGM SElect™ Kits FGA Identifiler® & NGM SElect™ Kits & Identifiler® Series Kits D21S11 Identifiler® & NGM SElect™ Kits D8S1179 Identifiler® & NGM SElect™ Kits VWA Identifiler® & NGM SElect™ Kits D16S539 Identifiler® & NGM SElect™ Kits TH01 Identifiler® & NGM SElect™ Kits D3S1358 Identifiler® & NGM SElect™ Kits AMEL Identifiler® & NGM SElect™ Kits HIGHLY RECOMMENDED LOCI D2S1338 Identifiler® & NGM SElect™ Kits (1) TPOX Identifiler® Kit D19S433 Identifiler® & NGM SElect™ Kits (2) D22S1045 NGM SElect™ Kit DYS391 Yfiler® Kit (3) SE33 NGM SElect™ Kit10/2/2012 | E Life Technologies™ PENTA © 22 (4) PENTA D
  • 23. Customer Feedback “With so many loci, isn’t “Our lab is dealing with more low level there a risk of losing trace and touch type samples, and analysis sensitivity and having more can be a nightmare, especially with drop out?” mixtures. With even more loci, I think that difficulty could increase exponentially.” “Not sure I see why you would want the additional Y marker. You “We are already facing big backlogs and already have Amelogenin and the budget shortfalls. Having to purchase Y marker in the quant kits.” and implement new instrument platforms is an enormous additional hurdle.”*Source: 2011 HID Future Trends in Forensic DNA Analysis Technology Seminar Series, Technical Lead Mtgs.10/2/2012 | © Life Technologies™ 23
  • 24. Development of a New STR Multiplex Project Goals  Maximize information recovery from challenging samples − Generate the most robust and discriminating kit available to the forensic community  Leverage new dyes to expand available space and incorporate additional markers − Meet the objectives of the CODIS Core Loci Working Group, ENFSI/EDNAP Requirements, and other international data- sharing initiatives10/2/2012 | © Life Technologies™ 24
  • 25. Kit Design: Challenges  Challenges associated with multiplexing large numbers of small amplicon STR loci − Insufficient space to accommodate large numbers of similarly-sized amplicons in the current 5-dye format − Limitations regarding the number of mobility modifiers that can be used to space out the amplicons − Expanded marker ranges limit space availability between adjacent markers  Challenges associated with balancing the needs of casework and databasing applications − Efficiency vs. robustness − High throughput vs. low-medium throughput − Expert system vs. manual analysis regimes10/2/2012 | © Life Technologies™ 25
  • 26. GlobalFiler™ Kits: Project Checklist Single multiplex that meets both international data-sharing requirements and the needs of forensic laboratories Separate kits for database and casework applications Compatible with validated instrument platform Incorporate all loci listed by the CODIS Core Loci Working Group as “Required” and “Recommended,” which include the required loci of the ESSL Maximize concordance with established databases and previously analyzed samples by maintaining current Identifiler®/NGM SElect™ Kit primer sequences Incorporate a Y-STR marker Maintain current amplicon size range of less than 460bp Incorporate additional alleles into the allelic ladder Optimize buffer system to maximize performance Optimize thermal cycling conditions to maximize sample processing efficiency10/2/2012 | © Life Technologies™ 26
  • 27. Kit Development Strategy The GlobalFiler™ Kits GlobalFiler™ GlobalFiler ™ Express PCR Amplification Kit PCR Amplification Kit A high performance, 6-dye An ultra-high throughput, chemistry for casework 6-dye chemistry for single samples source samples Optimized for use with the 3500/3500xL10/2/2012 | © Life Technologies™ 27
  • 28. Global Databases: Locus Overview Global- CODIS Global- CODIS Locus Europe CODIS Locus Europe CODIS Filer™ Proposed Filer™ Proposed *TPOX X X X D2S1338 X X X X CSF1PO X X X D19S433 X X X X D5S818 X X X D12S391 X X X D7S820 X X X D1S1656 X X X D13S317 X X X D2S441 X X X FGA X X X X D10S1248 X X X vWA X X X X *D22S1045 X X X D3S1358 X X X X *SE33 X X X D8S1179 X X X X DYS391 X X D18S51 X X X X *Recommended loci D21S11 X X X X TH01 X X X X All autosomal loci contained in either the Identifiler® or NGM SElect™ Kits D16S539 X X X X10/2/2012 | © Life Technologies™ 28
  • 29. Why Include Y?  Amelogenin Y null alleles occur − Additional Y testing may be required to confirm sample gender p q Amelogenin Y-indel/DYS391  Additional gender identification markers are on the long arm of the Y chromosome to minimise the risk of a double deletion  DYS391 is not a very polymorphic locus − YHRD data: 50% of haplotypes show allele 10; 2 null alleles reported; 0.01% duplication events (11 total) in >700 different worldwide population groups (ref: STRbase) − DYS391 is a stable locus with a relatively narrow allele range  Y-indel is short therefore will still perform even if DYS391 drops out in degraded samples10/2/2012 | © Life Technologies™ 29
  • 30. The GlobalFiler™ Kits: Multiplex Configuration10/2/2012 | © Life Technologies™ 30
  • 31. The GlobalFiler™ Kits: Planned Ladder Improvements D13S317 D19S433 GlobalFiler™ Kit range: 5-16 GlobalFiler™ Kit range: 6-19.2 Identifiler® Kit range: 8-15 NGM SElect™ Kit range: 9-17.210/2/2012 | © Life Technologies™ 31
  • 32. The GlobalFiler™ Kits: Allelic Ladder Use of mobility modifiers allows use of expanded ladder ranges whilst still maximising interlocus spacing10/2/2012 | © Life Technologies™ 32
  • 33. Optimizing for Performance and Throughput Buccal on Bode Buccal Collector™Blood on FTA® CardBuccal on Copan FLOQSwab™  Buffer system for the GlobalFiler™ Express Kit optimised to deliver high quality results on a wide range of substrates  Buffer system for the GlobalFiler™ Kit will be optimised to maximise performance on challenging casework samples10/2/2012 | © Life Technologies™ 33
  • 34. GlobalFiler™ Kits: Project Checklist Single multiplex that meets both international data-sharing requirements and the needs of forensic laboratories Separate kits for database and casework applications Compatible with validated instrument platform Incorporate all loci listed by the CODIS Core Loci Working Group as “Required” and “Recommended,” which include the required loci of the ESSL Maximize concordance with established databases and previously analyzed samples by maintaining current Identifiler®/NGM SElect™ Kit primer sequences Incorporate a Y-STR marker Maintain current amplicon size range of less than 460bp Incorporate additional alleles into the allelic ladder Optimize buffer system to maximize performance Optimize thermal cycling conditions to maximize sample processing efficiency10/2/2012 | © Life Technologies™ 34
  • 35. Maximizing Discrimination D10S1248 D22S1045 D16S539 D8S1179 D3S1358 D13S317 D2S1338 D19S433 D1S1656 D12S391 Kit PI* CSF1PO D21S11 D7S820 D18S51 D5S818 D2S441 TPOX TH01 vWA SE33 FGA Identifiler® Kit X X X X X X X X X X X X X X X 2.6 x 10-17 NGM SElect™ Kit X X X X X X X X X X X X X X X X 1.12 x 10-20 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 10-26 * Preliminary calculations based on a US Caucasian Database  The GlobalFiler™ Kits will: − Deliver a new level of discrimination − Reduce the risk of adventitious matches − Facilitate international DNA profile comparisons10/2/2012 | © Life Technologies™ 35
  • 36. Software Support for the GlobalFiler™ Kits GeneMapper® ID-X Software v1.4  Enables 6-dye STR Analysis and mixture interpretation  New GlobalFiler™ Kit-specific data analysis files − Panel, bins and stutter ratios − Table/plot settings − Size standard − Population database − CODIS marker properties file  Spike detection bypass for samples and controls − User selectable; if the switch is disabled, SSPK/SPK will be set to NA − Some SPK PQV rules adjusted to accommodate the spike detection switch10/2/2012 | © Life Technologies™ 36
  • 37. Software Support for the GlobalFiler™ Kits 3500 Data Collection Software v2.0 Updates  The software will warn the user (instead of a hard stop) if the polymer, anode or cathode buffer has expired − User can continue the run without changing the reagent, but the system will record the override − User will have the option to suppress future warnings  The software checks to make sure expiration dates (shelf-life and on-instrument-life) have not passed for the following: − Polymer > Expiration Date: Hard Stop  Warning Reagent and consumable lifetime > More than 7 days on instrument: Warning minimum performance guarantees are − Anode Buffer designed to promote the production of high > Expiration Date: Hard Stop  Warning quality data and minimize downtime across > More than 7 days on instrument: Hard Stop many laboratory environments − Cathode Buffer > Expiration Date: Hard Stop  Warning > More than 7 days on instrument: Hard Stop10/2/2012 | © Life Technologies™ 37
  • 38. The GlobalFiler™ Kits: Key Workflow Improvements Time to Result  Identifiler® Plus and Identifiler® Direct Kit amplification times: ~2.5 hours  GlobalFiler™ Express Kit fast and direct amplification time for single source samples: ~40 minutes  GlobalFiler™ Kit amplification time for casework samples: ~80 minutes Information Content  The GlobalFiler™ Kits will deliver the highest discrimination value of any kit previously produced by Life Technologies  The expanded allelic ladder ranges will improve automated genotyping efficiency when compared to the Identifiler® and NGM SElect™ Kits  A 6-dye configuration enables us to maximize the number of loci <220 bp enabling improved amplification from degraded DNA samples  Instrumentation and software enhancements will facilitate flexible and streamlined analysis10/2/2012 | © Life Technologies™ 38
  • 39. The GlobalFiler™ KitsApplication Development Insights and PerformanceOverviewHuman Identification, Life Technologies
  • 40. Agenda  The GlobalFiler™ Kits: General Development Insights − Kit Configuration − Primer Design − Allelic Ladder Expansion − Instrument Compatibility  Application-Specific Development and Testing Results − GlobalFiler™ Express Kit for Single-Source Sample Applications − GlobalFiler™ Kit for Casework Sample Applications  Summary10/2/2012 | © Life Technologies™ 40
  • 41. GlobalFiler™ Kit Configuration .10/2/2012 | © Life Technologies™ 41
  • 42. Designing the Optimal GlobalFiler™ Kit Configuration Version 1: The Starting Point - Identifiler® Plus & NGM™ Kits10/2/2012 | © Life Technologies™ 42
  • 43. Designing the Optimal GlobalFiler™ Kit Configuration Version 7: Incorporation of all ‘Required’ and ‘Recommended’ Loci10/2/2012 | © Life Technologies™ 43
  • 44. Designing the Optimal GlobalFiler™ Kit Configuration Version 13: Optimal Spacing between Loci10/2/2012 | © Life Technologies™ 44
  • 45. Maximizing Concordance  With the exception of TPOX, the GlobalFiler™ Kits utilize the same core primer sequences found in Identifiler® Plus/Identifiler® Direct and NGM SElect™ Kits − For TPOX and DYS391, the reverse primers were changed to optimize position and spacing within the multiplex In a population study of 1000 samples no discordance was observed in TPOX and DYS39110/2/2012 | © Life Technologies™ 45
  • 46. Minimizing False Homozygosity  Literature reports indicate instances of GlobalFiler™ Kit Loci w/New SNP- false homozygosity in the Identifiler®/NGM Specific Primers SElect™ Kits as a result of primer site SNP D3S1358 mutations vWA  Eight new SNP-specific primers have been D18S51 included in the GlobalFiler™ Kits to D19S433 minimize the occurrence of null alleles in future data sets TH01 − A series of SNP-specific primers already FGA exist in the Identifiler®/NGM SElect™ D5S818 Kits to account for other primer site mutations SE3310/2/2012 | © Life Technologies™ 46
  • 47. Maximizing Discrimination D10S1248 D22S1045 D16S539 D8S1179 D3S1358 D13S317 D2S1338 D19S433 D1S1656 D12S391 Kit PI* CSF1PO D21S11 D7S820 D18S51 D5S818 D2S441 TPOX TH01 vWA SE33 FGA Identifiler® Kit X X X X X X X X X X X X X X X 2.6 x 10-17 NGM SElect™ Kit X X X X X X X X X X X X X X X X 1.12 x 10-20 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 10-26 * Preliminary calculations based on a US Caucasian Database  The GlobalFiler™ Kits will: − Deliver a new level of discrimination − Reduce the risk of adventitious matches − Facilitate international DNA profile comparisons10/2/2012 | © Life Technologies™ 47
  • 48. An Unprecedented Power of Discrimination GlobalFiler™ Kit PI = 7.12 x 10-26 = 1 in 1.4 x 1025 Total Historical Human Population = 1.1 x 1011 The GlobalFiler™ Kits offer a power of discrimination 1014 times greater than the total population that has ever lived on Earth! Source - www.prb.org/10/2/2012 | © Life Technologies™ 48
  • 49. Enhanced Discrimination Power on Degraded Samples 1 2 10 STR Markers Under 220 bp 3 GlobalFiler™ Kits 4 5 10 mini-STRs < 220 bp 6 PI* = 9.2 x 10-12 7 8 9 10 Amplicon Mobility without mobility modifiers10/2/2012 | © Life Technologies™ 49 * Calculations based on a US Caucasian Database
  • 50. Enhanced Discrimination Power on Degraded Samples MiniFiler ™ Kit 8 mini-STRs < 220 bp PI* = 1.05 x 10-1010/2/2012 | © Life Technologies™ 50 * Calculations based on a US Caucasian Database
  • 51. Enhanced Discrimination Power on Degraded Samples GlobalFiler™ Kits 10 mini-STRs < 220 bp PI* = 9.2 x 10-12 MiniFiler ™ Kit 8 mini-STRs < 220 bp PI* = 1.05 x 10-10 Mini-STR Combination PI*= 5.73 x 10-2110/2/2012 | © Life Technologies™ 51 * Calculations based on a US Caucasian Database
  • 52. The GlobalFiler™ Kits: Gender Determination Improvements10/2/2012 | © Life Technologies™ 52
  • 53. The GlobalFiler™ Kits: Final Configuration Gender Determination Markers10/2/2012 | © Life Technologies™ 53
  • 54. What is an Indel?  Indels are short length polymorphisms, consisting of the presence (INsertion) or absence (DELetion) of a short sequence (1-50 nucleotides)  In the GlobalFiler™ Kits, an Indel marker on the Y chromosome was incorporated into the multiplex Deletion Insertion10/2/2012 | © Life Technologies™ 54
  • 55. Improved Accuracy of Gender Determination ♂ Y-indel position improves recovery in degraded samples10/2/2012 | © Life Technologies™ 55
  • 56. The GlobalFiler™ Kits: Allelic Ladder Improvements .10/2/2012 | © Life Technologies™ 56
  • 57. Expansion of the GlobalFiler™ Allelic Ladder Improving Genotyping Accuracy & Automation  Current Identifiler® and NGM SElect™ Kit allelic ladders do not contain all reported alleles  Utilizing literature reports and resources (e.g. STRbase), the genotyping accuracy of the GlobalFiler™ Kits was improved by: − Expanding the marker panels to accommodate new alleles − Adding new alleles to the allelic ladder − Adding new virtual bins to the bin file10/2/2012 | © Life Technologies™ 57
  • 58. GlobalFiler™ Kit Allelic Ladder Expansion D2S1338 D8S1179 GlobalFiler™ Kit range: 11-28 GlobalFiler™ Kit range: 5-19 Identifiler® Kit range: 15-28 Identifiler® Kit range: 8-1910/2/2012 | © Life Technologies™ 58
  • 59. The GlobalFiler™ Kits: Allelic Ladder 31 alleles added to 9 GlobalFiler™ Kit marker ladders New virtual bins added to improve genotyping efficiency10/2/2012 | © Life Technologies™ 59
  • 60. The GlobalFiler™ Kits: Instrument Compatibility & Calibration10/2/2012 | © Life Technologies™ 60
  • 61. GlobalFiler™ Kits: Instrument Compatibility GlobalFiler™ Kit GlobalFiler™ Express Kit (Casework Samples) (Single Source Samples) GeneAmp® 9700 Thermal Cycler with Silver or Gold- Veriti® Thermal Cycler Plated Silver 96-Well Block 3130 Series 3500 Series 3730* Genetic Analyzers Genetic Analyzers Genetic Analyzer GeneMapper® ID-X Software v1.410/2/2012 | © Life Technologies™ 61 * GlobalFiler™ Express Kit only
  • 62. A New 6-Dye Matrix Standard The GlobalFiler™ Kits utilize the DS36 (J6) Matrix Standard Set10/2/2012 | © Life Technologies™ 62
  • 63. Maximizing Color Balance for GlobalFiler™ Kit Data 3500xL 3130xl Variable binning settings for 3130 series instruments adjusted to gain optimal color balance10/2/2012 | © Life Technologies™ 63
  • 64. Maximizing Color Balance for GlobalFiler™ Kit Data 3500xL 3730 3730 settings required no adjustment10/2/2012 | © Life Technologies™ 64
  • 65. Streamlining Implementation GlobalFiler™ Kit Getting Started Bundles All components required to upgrade a 3130 or 3730 instrument in a single package Training / Chemistry Consumables Hardware Software Support 6- Dye GlobalFiler™ 6- Dye Data Installation Express kit Polymer Collection (SW upgrade, Software data migration, Dell XE calibration) 6-Dye Matrix computer Standard (for win7 compatibility) GeneMapper® 6- Dye Array ID-X Software Verification Size Standard v1.410/2/2012 | © Life Technologies™ 65
  • 66. The GlobalFiler™ Kits: Application-Specific Developments10/2/2012 | © Life Technologies™ 66
  • 67. GlobalFiler™ Kit Comparison GlobalFiler™ Express Kit GlobalFiler™ Kit Optimized to overcome inhibitors Optimized to provide optimal sensitivity Master Mix found in unpurified single source blood and intracolor balance for casework and buccal samples samples PCR protocol optimized for high PCR protocol optimized for speed of PCR Protocol sensitivity and high performance on processing for single source samples casework samples Control DNA 007 2 ng/µL 0.1 ng/µL Primer Mix Identical Primer Sequences Allelic Ladder Identical Allelic Ladder10/2/2012 | © Life Technologies™ 67
  • 68. GlobalFiler™ Express PCR Amplification Kit High Throughput Chemistry for Single Source Sample Applications10/2/2012 | © Life Technologies™ 68
  • 69. GlobalFiler™ Express Kit: Design Considerations  Achieve direct amplification from a variety of single source sample types: − Buccal samples on FTA® Card (treated paper) − Buccal samples on Bode DNA Collector™ (un-treated paper) − Buccal samples on swabs − Blood samples on FTA® Card (treated paper) − Blood samples on non-FTA® paper substrates (un-treated paper)  Obtain a high 1st Pass success rate for all sample types  Produce high quality STR profiles for all sample types − Metrics = intralocus and intracolor balance  Reduce thermal cycling time as much as possible10/2/2012 | © Life Technologies™ 69
  • 70. GlobalFiler™ Express Kit: Workflow Concepts Workflows are consistent with the Identifiler® Direct and NGM SElect™ Express Kits Exact volumes of individual reagents are kit-specific10/2/2012 | © Life Technologies™ 70
  • 71. Optimizing Reaction Time for the GlobalFiler™ Express Kit  Protocols developed on Veriti® thermal cyclers using 100% ramp speed  Performance verified on the 9700 thermal cyclers using MAX mode PCR Conditions 95°C/ 1 min 94°C/ 3 sec 25-27 x 60°C/ 30 sec 60°C/ 8 min Identifiler® Direct NGM SElect™ GlobalFiler™ Kit Express Kit Express Kit Total Time 2 hr 35 min 45 min 40 min10/2/2012 | © Life Technologies™ 71
  • 72. Optimizing Primer Mix for the GlobalFiler™ Express Kit Blood on Blood on Buccal on Buccal on Buccal on Buccal on FTA® Untreated Bode Buccal FTA® Copan Omniswab Bloodstain Collector ™ 4N6FLOQSwab ™ Card10/2/2012 | © Life Technologies™ 72
  • 73. GlobalFiler™ Express Kit Population Study Success Rate Number of Off- Sample Type N PCR Cycle Number (175 RFU) Scale Profiles Blood on FTA® 200 25 100% 4 Blood on Bloodstain Card (903 Paper) 45 25 100% 0 Buccal on FTA® (EasiCollect™ Device) 56 26 96.4% 3 Buccal on Bode Buccal Colector™ 90 26 96.6% 0 Buccal on Copan 4N6FLOQSwab™ 46 24 95.7% 3 Intralocus Balance Intracolor Balance N=3552 N=1000 N=761 N=1545 N=774 Blood on Buccal Blood on Buccal on Buccal on Blood on Buccal on Blood on Buccal on Buccal on FTA® on FTA® Untreated Bode Copan FTA® FTA® Untreated Bode Copan Bloodstain Buccal FLOQSwab™ Bloodstain Buccal FLOQSwab™ Card Collector™ Card Collector™10/2/2012 | © Life Technologies™ 73
  • 74. GlobalFiler™ Express Kit Data Examples Buccal on Bode Buccal Collector™ Blood on FTA® CardBuccal on Copan 4N6FLOQSwabs™  The GlobalFiler™ Express Kit is optimized to deliver high quality results on a wide range of substrates10/2/2012 | © Life Technologies™ 74
  • 75. GlobalFiler™ PCR Amplification Kit High Performance Chemistry for Casework Sample Applications10/2/2012 | © Life Technologies™ 75
  • 76. GlobalFiler™ Kit: Casework Design Considerations Casework Kit Performance Requirements  High sensitivity for trace/low level DNA samples  Good intracolor balance for mixture samples  Sufficient tolerance to PCR inhibitors  Efficient thermal cycling conditions10/2/2012 | © Life Technologies™ 76
  • 77. Optimization of GlobalFiler™ Kit Reaction Conditions Optimized thermal cycling PCR Conditions conditions shorten amplification 95°C/ 1 min conditions by > 1 hr 94°C/ 10 sec 29 x 59°C/ 90 sec Greater flexibility of sample input 60°C/ 10 min volume to facilitate analysis of low level samples Identifiler® Plus & NGM SElect™ Kits GlobalFiler™ Kit Total Time 2 hr 25 min 1 hr 20 min10/2/2012 | © Life Technologies™ 77
  • 78. GlobalFiler™ Kit Performance Example: Sensitivity Greater sensitivity was observed with the GlobalFiler™ Kit compared to the Identifiler® Plus Kit GlobalFiler™ Kit Identifiler ®Plus Kit10/2/2012 | © Life Technologies™ 78
  • 79. GlobalFiler™ Kit Performance Example: Inhibitor Tolerance Humic Acid Inhibition Hematin Inhibition Blue: Identifiler® Plus Kit Red: GlobalFiler™ Kit The GlobalFiler™ Kit buffer has been optimized to deliver enhanced inhibition tolerance compared to the Identifiler® Plus Kit10/2/2012 | © Life Technologies™ 79
  • 80. GlobalFiler™ Kit Performance Examples 500 pg 1 ng Intracolor Balance (%) Intralocus Balance GlobalFiler™ Identifiler® GlobalFiler™ Identifiler® Kit Plus Kit Kit Plus Kit Blue – Identifiler® Plus Kit Red – GlobalFiler™ Kit  The GlobalFiler™ Kit maintains the Stutter Percentage (%) high levels of intracolor balance, intralocus balance and stutter percentage established with previous next generation kits such as the Identifiler® Plus Kit10/2/2012 | © Life Technologies™ 80
  • 81. GlobalFiler™ PCR Amplification Kit α-Testing Results10/2/2012 | © Life Technologies™ 81
  • 82. Prototype GlobalFiler™ Kit vs. Identifiler® Kit Test Site #1: T-Shirt Swab; Organic Extraction Identifiler® Kit: No Profile GlobalFiler™ Kit: Full Profile (39 alleles) Identifiler® Kit : 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)10/2/2012 | © Life Technologies™ 82
  • 83. Prototype GlobalFiler™ Kit vs. Identifiler® Kit Test Site #2: Cigarette Butt, Organic Extraction Identifiler® Kit: No Profile GlobalFiler™ Kit: Full Profile (41 alleles) Identifiler® Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)10/2/2012 | © Life Technologies™ 83
  • 84. Prototype GlobalFiler™ Kit vs. Identifiler® Plus Kit Test Site #2: Bone Sample; Organic Extraction Identifiler® Plus Kit: Partial Profile (21 alleles) GlobalFiler™ Kit: Partial Profile (28 alleles) Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)10/2/2012 | © Life Technologies™ 84
  • 85. Prototype GlobalFiler™ Kit vs. NGM SElect™ Kit Test Site #2: Cigarette Butt, Organic Extraction NGM SElect™ Kit: Partial Profile (18 alleles) GlobalFiler™ Kit: Full Profile (43 alleles) NGM SElect™ Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)10/2/2012 | © Life Technologies™ 85
  • 86. Prototype GlobalFiler™ Kit Baseline Quality 74 444 Read Region 300 Test Site #1 300 Test Site #2 300 Test Site #310/2/2012 | © Life Technologies™ 86
  • 87. GlobalFiler™ Kit Feature Summary GlobalFiler™ Express Kit GlobalFiler™ Kit Amplifies the 20 ‘required’ and 3 ‘recommended’ loci in the new CODIS Core Loci recommendations Amplifies 3 Y chromosomal markers to ensure accurate gender determination Improves genotyping accuracy and automation through expanded allelic ladders and virtual bin sets Compatible with the Veriti® & 9700 thermal cyclers and the 3500 & 3130 series genetic analyzers Developed specifically for the direct amplification Developed specifically for the amplification of of single-source samples casework samples Delivers rapid throughput on blood and buccal Delivers increased sensitivity, improved intralocus samples on treated paper, untreated paper and and intracolor balance, enhanced performance on swabs inhibited and degraded samples Amplification Time ~ 40 min Amplification Time ~ 80 min10/2/2012 | © Life Technologies™ 87
  • 88. Development of a Next Generation Global Multiplex  Go Faster! − Up to 5x faster amplification time than previous generation kits > GlobalFiler™ Express Kit: ~40 minutes; GlobalFiler™ Kit: ~80 minutes > Enables sample to answer in <2 hours for single source samples  Go Further! − 24 loci, including 10 mini-STRs, increasing discrimination power by up to 9 orders of magnitude − Enhanced buffer system for even better performance on inhibited samples  Go Global! − Contains all loci listed by the CODIS Core Loci Working Group as “Required” and “Highly Recommended,” including the required loci of the ESSL − Maximizes global database compatibility10/2/2012 | © Life Technologies™ 88
  • 89. Go Further!  900 year old bone!  Medieval Austrain, Agnes of Waiblingen was the daughter of Salian King Henry IV and was married to Leopold III, one of the most important Babenberg Margraves  She died in 1143 and is reported to have had 17 children10/2/2012 89
  • 90. Acknowledgements  R&D  Forensic Science Applications Dennis Wang, Lori Hennessy, Siddhita Jennifer Schroeder, Lisa Calandro, Jeff Gopinath, Wilma Norona, Matthew Sailus, Nicola Oldroyd Ludeman, Robert Lagace, Chien-Wei Chang, Chang Zhong  Quality Assurance Liwei Qi, Yurah Yen  Tech Transfer Larry Wang, Anthony Matthews, Vanessa  Marketing Chan, Linda Liu, Yenyoung Lei Ariana Wheaton, Jonathan Tabak, Shereen Darwish, Lisa Schade  Software Allan Minn, Gloria Lam, Sharada  Oligo Development Viyachander, Tony Hatch, Makesh Kumar, Sarah Ruhe, Kathy Wang, Hong Ji Ravi Gupta, Doug Greig, Babu Yama  UK Manufacturing  Product Management Catherine Duke Karen Cormier, Bob Kays, Larry Joe, Jacki Benfield, Guido Sandulli  Project Management Wendy Coffey, Linda Kauffman10/2/2012 | © Life Technologies™ 90
  • 91. Thank You © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Refer to product page on the Life Technologies website for Limited Use License. The content provided herein may relate to products that have not been officially released and is subject to change without notice.10/2/2012 | © Life Technologies™ 91