1Life Technologies™ |
Improving your Casework Workflow with a
Next Generation Quantification Solution
Allison Holt
R&D Sci...
2Life Technologies™ |
Quan%filer®	
  	
  Duo	
  Kit	
  
Quan%filer®	
  	
  Human	
  Kit	
  
Quan%filer®	
  	
  Y	
  Human	
  ...
3Life Technologies™ |
Next Gen Quantifiler® HP & Quantifiler® Trio Kits
More Accurate, Sensitive, and Specific
Higher	
  P...
4Life Technologies™ |
Next Gen Quantifiler® Kit Has Faster Protocol
50°C 2 min.
95°C 10 min.
95°C 15 sec
60°C 1 min
Quanti...
5Life Technologies™ |
Streamlined Workflow Solutions
•  Flexible options for DNA standard curves decrease
reaction setup t...
6Life Technologies™ |
Multicopy Targets
Multicopy Targets selected:
1.  Maximum sensitivity
2.  Increased reproducibility
...
7Life Technologies™ |
Prototype Sensitivity – Data Consistency
1.2
pg/µL
20
ng/µL
DNA (ng/µL)
Sensitive and
Reproducibl
e
8Life Technologies™ |
Prototype Sensitivity – Data Consistency
1.2
pg/µL 20 ng/
µL
DNA (ng/µL)
Sensitive and
Reproducibl
e
9Life Technologies™ |
Sensitivity
Sample 007 DNA
§  At 1.25 pg/µL, partial profile is obtained with NGM® Select and all
d...
10Life Technologies™ |
Sensitivity
Sample 9947A DNA
§  At 1.25 pg/µL, partial profile is obtained with NGM® Select and al...
11Life Technologies™ |
Internal Positive Control
Correlates to Next Generation STR Kits
§  Master mix and primers enginee...
12Life Technologies™ |
Internal Positive Control
Correlates to Next Generation STR Kits
CT Shift Due to Hematin Inhibition...
13Life Technologies™ |
Improved Performance
Male/Female Mixtures
§  Male DNA detected with increasing amounts of backgrou...
14Life Technologies™ |
Multiple Targets
Balanced and Consistent
0.001
0.010
0.100
1.000
10.000
100.000
1:0 1:800 1:1000 1:...
15Life Technologies™ |
Quality Index (QI)
Comparison of Large Autosomal (LA) Target to Small Autosomal (SA) Target
• Diffe...
16Life Technologies™ |
Light Degradation
QI = 2.6
26 alleles (full profile)
Medium Degradation
QI = 23
16 alleles
High Deg...
17Life Technologies™ |
Blood
QI = 0.95
Sexual assault trace
QI = 4.5
Saliva on fabric
QI = 31.3
Saliva on fabric
QI = 115
...
18Life Technologies™ |
HID RT v1.2 Software
19Life Technologies™ |
Setup a Run
Define Samples, Sample Type and View Targets
Standards, targets, run method and
experim...
20Life Technologies™ |
Setup a Run
Assign Samples – Plate Layout view
21Life Technologies™ |
Data Analysis
Analysis Summary – QC standards to samples
22Life Technologies™ |
Data Analysis
Analysis Summary
§  Customizable View
designed to streamline
data review
§  Overvie...
23Life Technologies™ |
STR Amplification Set-up Tool
Dilu4on	
  Sengs	
  
STR	
  Reac4on	
  Sengs	
  
24Life Technologies™ |
STR Amplification Set-up Tool
§  Select the AmpFℓSTR® Kit(s)
for downstream analysis from
the STR ...
25Life Technologies™ |
Lab Configurable Software Solutions
§  Default Quantifiler ® and AmpFℓSTR® Templates can be optimi...
26Life Technologies™ |
Be Part of the Change!
Contact your local Human Identification support and sales representative
Bec...
27Life Technologies™ |
Acknowledgement
§  R&D:
−  Robert Green, Allison Holt, Mary Ma,
Pius Brzoska, Emanuel Langit,
Shar...
28Life Technologies™ |
Thank You!!
	
  
©	
  2013	
  Life	
  Technologies	
  Corpora%on.	
  All	
  rights	
  reserved.	
  ...
29Life Technologies™ |
Streamlined Workflow Solutions
8	
  Standards	
  
Manual	
  Run	
  
setup	
  
90	
  min	
  Amp	
  
...
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Improving your Casework Workflow with a Next Generation Quantification Solution

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Improving your Casework Workflow with a Next Generation Quantification Solution presentation given at the 2013 Life Technologies Forensic Seminar Series

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Improving your Casework Workflow with a Next Generation Quantification Solution

  1. 1. 1Life Technologies™ | Improving your Casework Workflow with a Next Generation Quantification Solution Allison Holt R&D Scientist
  2. 2. 2Life Technologies™ | Quan%filer®    Duo  Kit   Quan%filer®    Human  Kit   Quan%filer®    Y  Human  Kit Three  kits  –  for  different  applica4ons     Quan4fies  DNA  from  wide  variety  of  sources   Works  with  wide  variety  of  DNA  input   Sample  Extrac4on  chemistry  compa4ble   Quantifiler® Kits Product Family Quan%filer®    HP  Kit   Quan%filer®    Trio  Kit   HID  RT  v1.2  So=ware Two  new  Quan4filer®  kits  in  2013   +   Flexible  workflow  soLware   Integrate  with  Next  Genera4on  STR  Kits    
  3. 3. 3Life Technologies™ | Next Gen Quantifiler® HP & Quantifiler® Trio Kits More Accurate, Sensitive, and Specific Higher  Performance  with  Challenging  Samples   •   Improved  performance  with  low-­‐ra4o  male/female  mixture  samples     •   IPC  op4miza4on  to  match  performance  of  next  genera4on  STR  kits   •   Gain  key  informa4on  on  sample  quality  prior  to  PCR  amplifica4on   Enhanced  Workflow  &  Data  Analysis   •   Flexible  protocols  for  improved  sample  throughput   •   Improved  consistency  and  homogeneity  of  DNA  standard   •   Customized  workflow  templates    plus  data  analysis  and  STR  setup  tools   Fast  &  Accurate  Decision  Tool   •   Shorter  4me  to  results  compared  to  original  quan4fica4on  kits   •   Increased  assay  sensi4vity  and  accuracy   •   Solid  decision  making  for  forensic  casework  sample  processing  pathways   Fall 2013 Take  Control  of  your  Workflow  
  4. 4. 4Life Technologies™ | Next Gen Quantifiler® Kit Has Faster Protocol 50°C 2 min. 95°C 10 min. 95°C 15 sec 60°C 1 min Quantifiler  ® Human or Duo Quantifiler  ® Human Plus or Trio 95°C/2 min 95°C/ 9 sec 60°C/ 30 sec Sample *Protocol & thermal cycling conditions are approximate Amp Time ~ 90 min Amp Time 60 min*
  5. 5. 5Life Technologies™ | Streamlined Workflow Solutions •  Flexible options for DNA standard curves decrease reaction setup time and more samples per plate if a reduced number of standards are used •  Preconfigured Quantifiler ® Templates streamline sample setup – simply add/import sample names •  Decreased Cycle Amplification Time for faster time to result •  Integrated STR setup tools enable faster STR setup
  6. 6. 6Life Technologies™ | Multicopy Targets Multicopy Targets selected: 1.  Maximum sensitivity 2.  Increased reproducibility 3.  Awareness of homogeneity in populations •  Extensive bioinformatics analysis •  Tested with population database samples
  7. 7. 7Life Technologies™ | Prototype Sensitivity – Data Consistency 1.2 pg/µL 20 ng/µL DNA (ng/µL) Sensitive and Reproducibl e
  8. 8. 8Life Technologies™ | Prototype Sensitivity – Data Consistency 1.2 pg/µL 20 ng/ µL DNA (ng/µL) Sensitive and Reproducibl e
  9. 9. 9Life Technologies™ | Sensitivity Sample 007 DNA §  At 1.25 pg/µL, partial profile is obtained with NGM® Select and all detectors in Quantifiler® Trio give positive assay results. §  At 1.25 pg/µL, autosomal target CTs are ~36. Quantifiler® Results STR Results
  10. 10. 10Life Technologies™ | Sensitivity Sample 9947A DNA §  At 1.25 pg/µL, partial profile is obtained with NGM® Select and all detectors in Quantifiler® Trio give positive assay results. Quantifiler® Results STR Results
  11. 11. 11Life Technologies™ | Internal Positive Control Correlates to Next Generation STR Kits §  Master mix and primers engineered to correlate better with next generation STR chemistries §  Inhibitor tolerance is 5X higher then Quantifiler® Duo -2 0 2 4 6 8 10 12 14 16 0 20 40 60 80 100 120 CTShift(CTSample-CTCleanDNA) Humic Acid Concentration (ng/uL) CT Shift Due to Humic Acid Inhibition Autosomal Y Target IPC •  007 at 100 pg/µL •  Inhibitor Concentrations in the Quantifiler® Assays •  20-100 ng/µL humic acid •  Identifiler® Plus Reactions with 10 µL sample volume •  NOTE: multiply 5x inhibitor concentration of quantitation reaction to obtain the relative inhibitor concentration for STR reactions Identifiler® Plus Results (Alleles detected) Full 23/29 No profile
  12. 12. 12Life Technologies™ | Internal Positive Control Correlates to Next Generation STR Kits CT Shift Due to Hematin Inhibition -2 0 2 4 6 8 10 12 14 16 0 25 50 75 100 125 150 175 Hematin Concentration (uM) CTShift(CTSample-CTCleanDNA) Autosomal Y Target IPC Identifiler® Plus Results (Alleles detected) Full Profile No profile •  007 at 100 pg/µL •  Inhibitor Concentrations in the Quantifiler® Assays •  50–150 µM hematin •  Identifiler® Plus Reactions with 10 µL sample volume •  NOTE: multiply 5x inhibitor concentration of quantitation reaction to obtain the relative inhibitor concentration for STR reactions §  Master mix and primers engineered to correlate better with next generation STR chemistries §  Inhibitor tolerance is 5X higher then Quantifiler® Duo
  13. 13. 13Life Technologies™ | Improved Performance Male/Female Mixtures §  Male DNA detected with increasing amounts of background female DNA §  IPC Ct remained constant (26.3- 26.7) 0 0.5 1 1.5 2 2.5 3 0:1 1:0 1:1 1:5 1:10 1:20 Quantity(ng/uL) Ratio (male:female) Y Target Consistency Samples have 100 pg/uL Male DNA Y Target Autosomal Target
  14. 14. 14Life Technologies™ | Multiple Targets Balanced and Consistent 0.001 0.010 0.100 1.000 10.000 100.000 1:0 1:800 1:1000 1:1200 1:1400 Mean  Quant  Value  (ng/uL)  N  =  3 Mixture  Sample SA LA Y • Well balanced targets with non-degraded DNA • Y target remains constant in background of high female DNA
  15. 15. 15Life Technologies™ | Quality Index (QI) Comparison of Large Autosomal (LA) Target to Small Autosomal (SA) Target • Difference between the two targets assists users in identifying possible degradation 0.0001   0.0010   0.0100   0.1000   1.0000   10.0000   100.0000   Ctrl   Low   Med   Hi   DNA  Quan%ty  (ng/uL)  by  Target   DNA  Degrada%on  Condi%on   Small  Autosomal   Large  Autosomal  
  16. 16. 16Life Technologies™ | Light Degradation QI = 2.6 26 alleles (full profile) Medium Degradation QI = 23 16 alleles High Degradation QI = 300 6 alleles AmpFℓSTR® NGM™ PCR Amplification Kit Quality Index (QI) Artificially degraded human genomic DNA
  17. 17. 17Life Technologies™ | Blood QI = 0.95 Sexual assault trace QI = 4.5 Saliva on fabric QI = 31.3 Saliva on fabric QI = 115 NGM Select™ Kit profiles Quality Index (QI) Casework samples (not artificially degraded) External test site data Assists user in deciphering inhibition vs. degradation
  18. 18. 18Life Technologies™ | HID RT v1.2 Software
  19. 19. 19Life Technologies™ | Setup a Run Define Samples, Sample Type and View Targets Standards, targets, run method and experiment properties have been pre-defined for each Quantifiler® template
  20. 20. 20Life Technologies™ | Setup a Run Assign Samples – Plate Layout view
  21. 21. 21Life Technologies™ | Data Analysis Analysis Summary – QC standards to samples
  22. 22. 22Life Technologies™ | Data Analysis Analysis Summary §  Customizable View designed to streamline data review §  Overview of standard curve, NTC and unknown sample quality flags −  View samples that meet/do not meet defined threshold §  Hyperlinks to flagged samples for quick data review 1 2 3
  23. 23. 23Life Technologies™ | STR Amplification Set-up Tool Dilu4on  Sengs   STR  Reac4on  Sengs  
  24. 24. 24Life Technologies™ | STR Amplification Set-up Tool §  Select the AmpFℓSTR® Kit(s) for downstream analysis from the STR Kit Library §  Each kit template is pre- populated with default reaction set-up parameters and dilution scheme (customizable) §  Software determines sample dilution and volume added to the STR reaction based on Quantifiler® results
  25. 25. 25Life Technologies™ | Lab Configurable Software Solutions §  Default Quantifiler ® and AmpFℓSTR® Templates can be optimized based on your workflow – simply add/import samples §  Default Analysis Settings can be defined based on your SOP −  Choose which Data Quality Flags you wish to use as well as threshold values −  Dilution Calculations – define overage, min/maximum volumes, dilution factors −  STR Reaction Setup – target input amounts, reaction component and sample volumes. §  Data Display and Export – define Table, Plot and Plate views and Export formats
  26. 26. 26Life Technologies™ | Be Part of the Change! Contact your local Human Identification support and sales representative Become a Test Site • Support our rigorous development process • Give us feedback and help us build the kits that meet your needs • Be the first to evaluate the Quantification kits with your challenging samples
  27. 27. 27Life Technologies™ | Acknowledgement §  R&D: −  Robert Green, Allison Holt, Mary Ma, Pius Brzoska, Emanuel Langit, Sharon Wootton, Julio Mulero §  Oligo −  Sarah Ruhe, Emma Duimstra, Hong Ji §  Product Management: −  Sheri Olson §  Forensic Science Applications: −  Jacki Benfield, Jeff Sailus, Jill Muehling §  Tech Transfer −  Ellen Kuo , Yenyoung Lei §  Project Management −  Ada Wong §  Manufacturing Sciences −  John Dektar, Yurah Yen, Ryo Hasegawa §  Marketing −  Shareen Fousekis, Jonathan Tabak §  Manufacturing −  Marilena Philippidou, Catherine Duke
  28. 28. 28Life Technologies™ | Thank You!!   ©  2013  Life  Technologies  Corpora%on.  All  rights  reserved.       The  content  provided  herein  may  relate  to  products  that  have  not  been  officially   released  and  is  subject  to  change  without  no%ce.     “FOR  FORENSIC  AND  PATERNITY  USE  ONLY”    
  29. 29. 29Life Technologies™ | Streamlined Workflow Solutions 8  Standards   Manual  Run   setup   90  min  Amp   Manual  STR   setup   Flexible   Standards   Op%ons   Streamlined   Run  setup    -­‐   Quantifiler®     Templates   ~60  min  Amp   Integrated,   Automated  STR   setup   Quantifiler® Trio and Human Plus Quantifiler® Duo and Human
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