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Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar

Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar



Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar presentation given at the 2013 Life Technologies Forensic Seminar Series

Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar presentation given at the 2013 Life Technologies Forensic Seminar Series



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    Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar Presentation Transcript

    • 1Life Technologies™ | Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit Kellie Fenesan Life Technologies
    • 2Life Technologies™ | Total  Time  to  Result  =  ~10-­‐58  hrs    Sample     Prepara9on   Quan9ta9on/   Normaliza9on   Amplifica9on   Detec9on   Analysis      Time  to  Result     (hrs/per  6-­‐16  samples)   1-­‐48   2   3.5   0.75   2-­‐3  Time  to  Result     (hrs/per  13  samples)   1-­‐3   2     1.5   0.55   1-­‐2   Total  Time  to  Result  =  ~6-­‐9  hrs   Dependent  on   sample  type   and  method   Improved Efficiency for Casework Samples
    • 3Life Technologies™ | Detection & Analysis Our Mission: Improve Efficiency. Increase Success Rates. Provide More Info. LIFE Offers Complete Integrated HID Workflow GeneMapper® ID-X Expert System Software Next Generation STR Kits 3500/3500xL Genetic Analyzer 3130/3130xl Genetic Analyzer Extraction Quantification Amplification 7500 Real-Time PCR Instrument Next Generation Quantifiler® Kits PrepFiler™ Forensic DNA Extraction kit Veriti® 96-Well Thermal Cycler GeneAmp® PCR System 9700 Cycler AutoMate Express™ Collection Copan 4N6FLOQSwab™ & NUCLEIC-CARD™ Screening ParaDNA® Converge™ Software DNA Workflow Kinship and Paternity Direct Search Familial Search NEW NEW NEW NEW Converge™ Software NEW NEW
    • 5Life Technologies™ | Which Profile Would You Rather Interpret? This One?
    • 6Life Technologies™ | Or This One?
    • 7Life Technologies™ | The GlobalFiler™ Kit: Going Further for Casework
    • 8Life Technologies™ | What is the GlobalFiler™ Kit? 1.  D.R.  Hares,  Expanding  the  CODIS  core  loci  in  the   United  States,  Forensic  Sci.  Int.  Genet.    6  (2012),   e52-­‐e54.     2.  D.R.  Hares,  Addendum  to  expanding  the  CODIS   core  loci  in  the  United  States,  Forensic  Sci.  Int.   Genet.  6  (2012),  e135.   The GlobalFiler™ Kits are 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 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   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   SE33   X   X   X  
    • 9Life Technologies™ | A short history of STR Kit Multiplexing Kit Name Release Date # of Loci # of Dyes* AmpFSTR® Blue 1996 3 2 AmpFSTR® Profiler® 1997 10 4 AmpFSTR® Identifiler® 2001 16 5 GlobalFiler™ 2012 24 6 *Includes size standard
    • 11Life Technologies™ | Multiplex Configuration Strategy
    • 12Life Technologies™ | 5-Dye Configuration Containing All Required and Highly Recommended Loci 1 2 3 4 Multiple loci > 400 bp à poor performance for degraded DNA à compromised sizing and resolution 1 2 3 4 5 6 7 8 Fewer Mini-STRs à poor performance for degraded DNA Primer Redesigns à less concordant
    • 13Life Technologies™ | The GlobalFiler™ Kits: 6-Dye Configuration 1 2 3 4 5 6 7 8 9 10 GlobalFiler™ Kit 10 Mini-STRs PI* = 9.2 x 10-12 MiniFiler ™ Kit 8 Mini-STRs PI* = 1.05 x 10-10 11 12 13 14 15 16 17 18 18 Mini-STRs Combination PI*= 5.73 x 10-21 97% of alleles in allelic ladder <400bp Only 2 loci require primer redesign* *  Preliminary  calcula.ons  based  on  a  US  Caucasian  Database   DYS391 Amel Y indel All gender markers located in the same dye channel for ease of interpretation
    • 14Life Technologies™ | •  Amelogenin  Y  null  alleles  can  occur   •  Addi9onal  Y  tes9ng  may  be  required  to  confirm  sample  gender     •  Addi9onal  gender  iden9fica9on  markers  are  on  the  long  arm  of  the  Y  chromosome   to  minimize  the  risk  of  a  double  dele9on   •  DYS391  is  a  stable  locus  with  a  rela9vely  narrow  allele  range     •  The  Y-­‐indel  is  very  short  therefore  more  likely  to  perform  even  if  DYS391  drops  out   in  degraded  samples   p q Amelogenin   Y-­‐indel/DYS391   Why Include Additional Y Markers?
    • 15Life Technologies™ | • Gender identification redundancy helps remove the need for laboratories to maintain a second gender confirmation test protocol • In addition, the Y-indel position (<90 bp) in the GlobalFiler™ Kit helps improve data recovery in degraded samples ♂ ♀ Improved Accuracy of Gender Determination
    • 17Life Technologies™ | Facts about the SE33 Locus §  The most discrimination power of any STR locus in commercial kits −  highest Hetobs (0.9353) −  lowest PI 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 - 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
    • 18Life Technologies™ | SE33 Locus – Ideal for Mixtures
    • 19Life Technologies™ | Overlay of SE33 Data from 4 individuals SE33 extremely valuable for mixture interpretation In this case, we have 4 donors and 7 peaks
    • 20Life Technologies™ | Maximizing Allele Calls: The GlobalFiler™ Kits Allelic Ladder New virtual bins added to improve genotyping efficiency 343 alleles 246 virtual bins 589 total alleles 31 alleles added to 9 GlobalFiler™ Kit marker ladders
    • 21Life Technologies™ | Achieving New Levels of 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 10-18 NGM SElect™ Kit X X X X X X X X X X X X X X X X 2.35 x 10-21 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 GlobalFiler™ Mini’s X X X X X X X X X X *9.20 x 10-12 * Preliminary calculations based on a US Caucasian Database §  The GlobalFiler™ Kits: −  Deliver a new level of discrimination −  Reduce the risk of adventitious matches −  Facilitate international DNA profile comparisons The GlobalFiler™ Kits offer a power of discrimination 1014 times greater than the total population that has ever lived on Earth!
    • 22Life Technologies™ | GlobalFiler™ Kits: Instrument Compatibility §  Supported Platforms §  3130/3130xL Genetic Analyzer* §  3730 DNA Analyzer (GlobalFiler™ Express Kit only) §  3500/3500xL Genetic Analyzer *3100 series may be upgraded to 3130
    • 23Life Technologies™ | Instrument Compatibility 3500xL 3130xl
    • 24Life Technologies™ | 6-Dye Configurations Chemistry/   Standards         GeneMapper®   ID-­‐X  So`ware   v1.4   Service   3730  DNA   Analyzer  /   31xx  Geneec   Analyzers   3500  Geneec   Analyzer   Data  Colleceon   So`ware   1   2   3   31xx/3730  only    All  Instruments    
    • 25Life Technologies™ | GlobalFiler™ Kit Upgrade Roadmap Product  Type   3500   3730   3130   3100   GeneMapper®  ID-­‐X  So`ware   v1.4   Data  Colleceon     So`ware  v4.0   N/A   6-­‐dye  Data  Colleceon   Module   N/A   Service  Call/Computer   N/A   Instrument  Upgrade   N/A   N/A   N/A  
    • 26Life Technologies™ | Application Specific Development & Testing
    • 27Life Technologies™ | Kit Development Strategy GlobalFiler™ PCR Amplification Kit GlobalFiler ™ Express PCR Amplification Kit The GlobalFiler™ Kits A high performance, 6-dye chemistry for casework samples An ultra-high throughput, 6- dye chemistry for single source samples
    • 28Life Technologies™ | 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 §  Maintain high species specificity §  Efficient thermal cycling conditions
    • 29Life Technologies™ | Decreased amplification time for casework samples Op9mized  thermal  cycling  condi9ons   shorten  amplifica9on  9me  to  ~80  mins   PCR  Condi9ons               29  x   Total  Time     Iden9filer®  Kit   ~3.5  hours   Iden9filer®  Plus  Kit  &   NGM  SElect™  Kit   ~2.5  hours   GlobalFiler™  Kit   ~80  mins   95°C/  1  min   94°C/  20  sec   59°C/  90  sec   60°C/  10  min  
    • 30Life Technologies™ | GlobalFiler™ Kit Test Site Data
    • 31Life Technologies™ | TS1   TS2   TS3   TS4   TS5   Extraceon   Method   Organic  or  EZ1   Organic   Magne9c   Bead   Qiagen   Inves9gator   Organic   Comparaeve   Kit   Iden9filer   Plus,  PP  16HS,     PP  Fusion   NGM  SElect   Iden9filer   Plus   Iden9filer   Plus   NGM  SElect   Thermal   Cycler   9700  Gold   Veri9   Veri9   9700  Gold   9700  Gold   CE   Instrument   3500   3500   3500xL   3500xL   3500   GlobalFiler™ Kit Test Site Overview
    • 32Life Technologies™ | 300 Test Site #1 GlobalFiler™ Kit Baseline Quality 300 300 Test Site #2 Test Site #3 Read  Region   74   444   Non-template Controls
    • 33Life Technologies™ | The  GlobalFiler™  Kit    accommodates  up   to  15  μL  of  sample  input  volume  to   facilitate  improved  performance  for   low  level  samples.   Reagent  Volumes     Master  Mix                    7.5  µL   Primer  Set                        2.5  µL   Sample  Input    ≤15.0  µL                                                          25.0  µL   Increased Sensitivity
    • 34Life Technologies™ | GlobalFiler™ Kit Performance Example: Sensitivity GlobalFiler™ Kit [DNA]     D8     D21     D7     CSF     D3     TH01     D13     D16     D2(S1)   D19     vWA     TPOX     D18     Amel     D5     FGA     D1   D10   D12   D2(S4)   D22   DY3   SE33   Yind   1  ng                                                                                                                                                                                                                                                                                                                                                                                                       500  pg                                                                                                                                                                                                                                                                                                                                                                                                     250  pg                                                                                                                                                                                                                                                                                                                                                                                                     125  pg                                                                                                                                                                                                                                                                                                                                                                                                     62.5  pg                                                                                                                                                                                                                                                                                                                                                                                                     31.25  pg                                                                                                                                                                                                                                                                                                                                                                                                     15.63  pg                                                                                                                                                                                                                                                                                                                                                                                                     Increased allele recovery with low input samples.
    • 35Life Technologies™ | Sensitivity Results <50pg of DNA using 10uL and 15uL Sample Input Volume TS  1   TS2   TS3   TS4   TS  5   46.88  pg   4   5   16   13   33   38   36   38   18   15   15   26   19   12   16   26   29   35   30   10   31.  25  pg   4   3   1   2   25   24   24   22   6   8   7   12   20   10   6   18   21   18   22   26   23.43  pg   4   2   4   4   12   21   15   12   3   11   7   3   4   5   6   8   10   17   12   19   15.62  pg   0   0   1   1   15   12   14   12   0   1   1   0   1   6   5   2   6   4   7   8   • Variability in allele recovery from site to site
    • 36Life Technologies™ | [DNA]  =  1.5  pg/uL   [DNA]  =  3.1  pg/  uL   N  =  4   Sample  Input  Volume   Increase  in   Recovery   Sample  Input  Volume   Increase  in   Recovery  10  uL   15  uL   10  uL   15  uL   Test  Site  1   0.5   3.5   7x   2.5   9.5   3.8x   Test  Site  2   13.25   15   1.11x   23.75   36.25   1.53x   Test  Site  3   0.5   6   12x   8.25   18.5   2.24x   Test  Site  4   3.5   5.75   1.64x   13.5   18.25   1.35x   Test  Site  5   6.25   14.5   2.32x   21.75   26   1.2x   Total   24   44.75   1.86x   69.75   108.5   1.56x   Allele Recovery: Extended Sample Volume Input Average alleles per replicate shown for each input volume • Test sites ran the same samples varying only the volume of DNA added to the reaction. • Adding an additional 5uL of DNA to the reaction delivered approximately 1.5 -2.0 times more alleles in low concentration samples
    • 37Life Technologies™ | GlobalFiler™ Kit Test Site Results NGM SElect™ Kit: Full Profile (26 alleles) GlobalFiler™ Kit: Full Profile (36 alleles) Cigarette Butt, Organic Extraction NGM SElect™ Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)
    • 38Life Technologies™ | GlobalFiler™ Kit Test Site Results Identifiler® Plus Kit: Partial Profile (21 alleles) GlobalFiler™ Kit: Partial Profile (28 alleles) Bone Sample; Organic Extraction Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)
    • 39Life Technologies™ | GlobalFiler ™ Kit: Full (40 alleles)Identifiler® Plus Kit: Partial (21 alleles) Door Handle Swab, Qiagen Investigator (310pg) Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit:175 RFU PAT (3500xL) GlobalFiler™ Kit Test Site Results
    • 40Life Technologies™ | GlobalFiler™ Kit Test Site Results NGM SElect™ Kit: Partial/Mixture (24 alleles) GlobalFiler™ Kit: Partial/Mixture (37 alleles) Underwear, Organic Extraction NGM SElect™ Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)
    • 41Life Technologies™ | Test Site Results: 900 Year Old Bone Sample §  Medieval  Austrian,  Agnes  of   Waiblingen  was  the  daughter   of  Henry  IV,  Holy  Roman   Emperor  and  was  married  to   Leopold  III,  one  of  the  most   important  Babenberg   Margraves,    who  ruled  the   Austrian  territory     §  Born  in  1072,  she  died  in   1143  (aged  71)  and  is   reported  to  have  had     >20  children    
    • 42Life Technologies™ | Partial Profiles: Alleles Recovered Using the GlobalFiler™ and Identifiler® Plus Kits 0   5   10   15   20   25   30   35   40   45   Fat  Trace   Swab  /310pg   Key  Swab/   60pg   Boqleneck   Swab  /  50pg   Drawer  Swab/ 60pg   Swab  on   Safe  /  50pg   Wheel  Swab  / 80pg   Cigareqe   Buq  /  40pg   Cigareqe   Buq  /  30pg   Cap  Swab  /   165  pg   Stone  /  Undet   No.  Alleles  Recovered   Pareal  Profile  Samples   GF  Par9al     ID+  Par9al   The increased robustness of the GlobalFiler™ Kit delivered 182% more alleles than the Identifiler ® Plus Kit with partial profiles • Deliver more information per sample • Increases the likelihood of a meaningful database match
    • 43Life Technologies™ | GlobalFiler™ Kit Configuration and Workflow - A change has been made to the configuration of the GlobalFiler™ Kit workflow to provide longer term expiration dating (18 months at -20ºC) - Kits will be received in the standard frozen condition. - Prior to first use, the users will: 1)  Thaw reagents (Standard Protocol) 2)  Add 83 uL of Master Mix Additive reagent into Master Mix tube. 3)  Invert the Master Mix tube 10 times. Mark the Master Mix tube with a + to indicate MM Additive was added. Discard Additive tube. 4)  Master Mix is ready for use and to be stored at 4ºC (Standard Protocol). Traditional Kit Configuration GlobalFiler™ Kit Configuration P M   M M   A d d   P M   M M   New   New   New  
    • 44Life Technologies™ | GlobalFiler™ Kit: Test Site Feedback “The baseline is perfect, no artifact was observed in negative controls. Sample profiles have showed good quality data with a good balance PHR.”   “The fact that we are able to use up to 15µL of DNA per PCR reaction improves the sensitivity … very interesting for the amplification of poor DNA samples (<50pg/ µL).”   “Having 50% of the PCR products with a size less than or equal to 220bp allows better amplification of degraded DNA. In addition, with a volume of 15µL of DNA per PCR reaction, we are in the best conditions for amplification of degraded DNA with an amount of DNA lower than100pg/µL.”   “Seems to withstand well to inhibitors…”  
    • 45Life Technologies™ | GlobalFiler™ Kit: Test Site Feedback “Thanks to the integration of DYS391 and the Y-indel we can no longer have any doubt concerning the analysis of the Y chromosome in the case of amelogenin mutation.”   “Using a 6th dye allows to separate the markers analyzed and thus avoid allele overlap between neighboring markers.”   “The use of this kit should reduce the sample rate that has to be re-amplified whether with the same PCR kit or with another kit, while improving performance…”   “PCR  dura9on  (1:20  vs.   2:30  with  Iden9filer®  Plus)   is  also  an  advantage   especially  in  the  context  of   custody…”  
    • 46Life Technologies™ | 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 §  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
    • 47Life Technologies™ | Thank  You   ©  2013  Life  Technologies  Corpora9on.  All  rights  reserved.  The   trademarks  men9oned  herein  are  the  property  of  Life  Technologies   Corpora9on  or  their  respec9ve  owners.       The  content  provided  herein  may  relate  to  products  that  have  not  been   officially  released  and  is  subject  to  change  without  no9ce.     “FOR  FORENSIC  AND  PATERNITY  USE  ONLY”   http://www.slideshare.net/Lifetech_HID http://www.lifetechnologies.com/globalfiler