hid validation the 3500 series genetic analyzers · hid validation the 3500 series genetic...

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HID University Seminar Series 2010

HID Validation The 3500 Series Genetic Analyzers

2 Proprietary & Confidential

Outline

•  Scope •  Materials and Reagents

•  Validation Elements •  Selection of Validation Data

•  Additional Sources of Validation Information •  User Bulletin


Validation Scope Hardware and Reagents

•  Instruments •  (3) 8 Capillary Instruments (3500) •  (3) 24 Capillary Instruments (3500xL)

•  Size Standard • GeneScan™ 600 LIZ™ Size Standard v2.0 • GeneScan™ 500 ROX™ Size Standard

•  Profiler Plus™ / Cofiler™ do not use normalization

•  Polymer and Array • POP-4™ and 36 cm Capillary Array


Validation Scope Sample Summary

Test Name Samples Input Replicates

Genotype Concordance and Reproducibility

• 40 male and 42 female gDNA samples • 4 racial groups • Controls

1-2 ng total DNA • 1 replicate per sample • 2 injections

Sizing Precision and Accuracy

Allelic ladder 1 µL per well • See below

Sensitivity and Normalization

• 007 control DNA • 3 gDNA samples

• STR: 0.125ng, 0.5ng, 1ng, 6ng

• 5 replicates for DNA samples • 3 replicates for NTC • 4 injections • Minifiler: 0.125ng,

0.25ng, 0.5ng, .75ng

Mixture Analysis • 4 pairs of male and female gDNA • Mixture ratios • 1:0, 1:1, 1:3, 1:5, 1:9, and 0:1

• Recommended concentrations per kit

1 replicate per sample

Resolution • Allelic Ladder • Research material

• 1 µL per well 24 replicates


Validation Scope AmpFℓSTR™ Kits

•  Identifiler™

•  Minifiler™

•  SGM Plus™ •  Yfiler™ •  Sinofiler™ •  SEfiler Plus™ •  Profiler Plus™ •  Cofiler™ •  Identifiler™ Direct •  Identifiler™ Plus •  NGM™


Precision and Accuracy


Precision and Accuracy Example Configurations for Standard Deviation

Within Injection - 3500

8 data points per mean 12 injections per plate 12 columns 3 Instruments

12 data points per mean 4 injections per plate 8 rows 3 Instruments

Across Injections – 3500xL


3500 Sizing Precision Study: Within an Injection of Identifiler® Allelic Ladder


3500 Sizing Precision Study: Across Multiple Injections of Identifiler® Allelic Ladder Samples

96 Replicates of Identifiler® Allelic Ladder

3500 -12 Injections 3500xL - 4 Injections


Introduction to Normalization


Sources of CE Variation (3100)

•  Numerous optical components, capillary array

•  Electrophoresis, injection

•  Sample and sample preparation

Raman Intensity, 3100file: INSTRUMENT DATA Mod2B.xls

y = -2.7202x + 6042.8R2 = 0.0059

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 20 40 60 80 100 120 140Instrument

A/D

Units

Series1Linear (Series1)

median= 6026.50Stdev = 808.54Average = 6054.11Stdev/ average = 0.13

Almost 2-fold difference in signal from over 100 instruments from the factory floor


Normalization: What is it? •  This feature attenuates

signal variations associated with instrument, capillary array, sample salt load, and injection variability among capillaries and instruments

•  Normalization can be applied during primary analysis of the data

Before normalization After normalization

3500-Series Normalization Feature


•  Normalization Target (NT) •  The expected average RFU for eleven peaks in the GeneScan 600

LIZ® v2 size standard used for normalization

•  Normalization Factor •  Average peak height of the subset of peaks in the GeneScan 600

LIZ® v2 size standard used for normalization divided by the Normalization Target

•  Normalization Factor Threshold •  The passing range for Normalization Factor (default range is 0.3 to

3.0)

Normalization: Definitions

3500-Series normalization calculations


No NormFactory only

Int Std onlyFactor+IntStd

Injection to Injection

Cap to Cap

Within

Instrument to Instrument

Total

39%

33%

25%

14%

31%

24% 24%

13%

20%19%

5%5%

12%11%

4%4%3%

3%

0%0%

0%

5%

10%

15%

20%

25%

30%

35%

40%

%CV

Levels of Normalization

Sources of Variation

Instrument to Instrument largest source of variation

Internal standard

Normalization greatest reduction in variation

Sources of variation by Normalization Method


Sensitivity and GeneScan™ 600 LIZ™ Size Standard v2.0


Sensitivity Study: Average Peak Height across a Range of DNA Input Amounts AmpFℓSTR™ MiniFilerTM PCR Amplification Kit

Normalization Method Factory Only Factory + Internal Std

0.75ng0.5ng0.25ng0.125ng

20000

15000

10000

5000

0

Conc. 0.75ng0.5ng0.25ng0.125ng

20000

15000

10000

5000

0

H1126

Ave

rage

Pea

k H

eigh

t

H1152

H1161 OO7

offon

Normalization

Boxplot of Average Peak Height

Panel variable: Sample

10000

5000

15000

0 0.125ng 0.25ng 0.50ng 0.75ng

10000

5000

15000

0

0.125ng 0.25ng 0.50ng 0.75ng Genomic DNA #2

Control DNA Standard 007

Genomic DNA #1

Genomic DNA #3

Peak

Hei

ght


Normalization onoff

14000

12000

10000

8000

6000

4000

2000

0

Ave

rage

Pea

k H

eigh

t

Boxplot of Average Peak HeightConc. = 1ng

2000

4000

8000

10000

6000

12000

14000

Internal Standard Normalization

2000 2000

Disabled Enabled

Peak

Hei

ght

Sources of CE Variation (3100) Sources of CE Variation (3100) Sensitivity Study: Average Peak Height by Dye for 1ng DNA input AmpFℓSTR™ Identifiler™ PCR Amplification Kit


Size Standard Peak Height Consistency Study: Five AmpFℓSTR™ PCR Amplification KitsGeneScan™ 600 LIZ™ Size Standard v2.0

6000

5000

4000

3000

2000

1000

0

Lot 1 Lot 2 Lot 3

GS600 Peak Height

Identifiler™ Identifiler™ Direct Identifiler™ Plus MinifilerTM NGMTM

7000

GS600 Lot Number

Peak

Hei

ght


% CV

Size Standard Peak Height Consistency · 6 instruments · 3 lots of size standard

Size Standard Peak Height Consistency Study: %CV for 5 Different AmpFℓSTR® Kits GeneScan™ 600 LIZ® Size Standard v2.0


Internal Standard Normalization: Injection to Injection Peak Height Consistency GeneScan™ 600 LIZ™ Size Standard v2.0

% CV

Normalization Method Factory Only Factory + Internal Std

40

30

20

10

0 Lot 1 Lot 2 Lot 3

Size Standard Peak Height Consistency · 6 instruments · 3 lots of size standard


Resolution and Baseline Evaluation


Resolution

•  SEfiler Plus™ (SE33 triplet) and Identifiler™ (TH01 9.3/10) Allelic Ladders

•  160 injections per instrument on 6 instruments

•  Research STR markers greater than 300 base pairs were also developed with single base pair differences (data not shown)


Baseline Noise - Developmental Evaluation G5 Average Noise (10x SD)

0

20

40

60

80

100

120

140

160

180

200

FAM VIC NED PET LIZ TOTAL

10x

Ave

rage

Std

. Dev

.

Data generated during the Sensitivity experiment was then used to confirm the level of pull up and the peak amplitude threshold setting.

175 RFU


Pull-up 360 Samples at 6 ng template

5.0%

20000

4000

8000

12000

16000

24000


Additional Studies and Validation Documentation


Identifiler™ Direct

•  82 gDNA, 25 cycles •  Three 3500xl and three 3500 instruments. •  Mother plate split into 6 daughter plates.

8000

8000

8 Capillary 3500

24 Capillary 3500xL

14000

14000

0

0


Additional Studies

•  Genotype Concordance •  100% Concordant with 3130xL

•  GeneMapper™ ID-X v1.2 Evaluation • Normalization and RFID documentation functionality performed as

expected • Average Mx values obtained in the Mixture Study were similar to the

3130xl


Validation Documentation

•  AAFS and ISHI Posters •  User Bulletin containing developmental validation •  www.thermofisher.com/3500hid


THANK YOU!!

Ariana Wheaton Senior Market Development Manager Thermo Fisher Scientific Email: [email protected]

© 2016 Thermo Fisher Scien3fic Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scien3fic and its subsidiaries unless otherwise specified. Windows is a trademark of MicrosoG Corpora3on. Products described herein are intended for forensic or paternity use only. Not for use in diagnos3cs procedures. The content provided herein may relate to products that have not been officially released and is subject to change without no3ce.

Na Parra Product Manager Thermo Fisher Scientific Email: [email protected]

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