Thermo Fisher Scientific HID Validation: The 3500 Series Genetic Analyzers Owner's manual

HID Validation:

The 3500 Series Genetic Analyzers

2

HID Validation:

The 3500 Series Genetic Analyzers

Outline

•Scope

•Materials and Reagents

•Validation Elements

•Selection of Validation Data

•Additional Sources of Validation Information

•User Bulletin

3

HID Validation:

The 3500 Series Genetic Analyzers

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

4

HID Validation:

The 3500 Series Genetic Analyzers

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

5

HID Validation:

The 3500 Series Genetic Analyzers

Validation Scope

AmpFℓSTR®Kits

•Identifiler®

•Minifiler™

•SGM Plus®

•Yfiler®

•Sinofiler™

•SEfiler Plus™

•Profiler Plus®

•Cofiler®

•Identifiler®Direct

•Identifiler®Plus

•NGM™

Precision and Accuracy

7

HID Validation:

The 3500 Series Genetic Analyzers

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

8

HID Validation:

The 3500 Series Genetic Analyzers

3500 Sizing Precision Study:

Within an Injection of Identifiler®Allelic Ladder

9

HID Validation:

The 3500 Series Genetic Analyzers

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

11

HID Validation:

The 3500 Series Genetic Analyzers

Sources of CE Variation (3100)

•Numerous optical

components,

capillary array

•Electrophoresis,

injection

•Sample and

sample

preparation

Raman Intensity, 3100

file: INSTRUMENT DATA Mod2B.xls

y = -2.7202x + 6042.8

R

2

= 0.0059

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

020 40 60 80 100 120 140

Instrument

A/D Units

Series1

Linear (Series1)

median= 6026.50

Stdev = 808.54

Average = 6054.11

Stdev/ average = 0.13

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

12

HID Validation:

The 3500 Series Genetic Analyzers

Normalization: What is it?

•This feature normailzes

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

13

HID Validation:

The 3500 Series Genetic Analyzers

•Normalization Target (NT)

•The expected average RFU for eleven peaks in the

GeneScan 600 LIZ®v2 size standard used for normalization

•Normalization Factor

•Normalization Target divided by the Average peak height of

the subset of peaks in the GeneScan 600 LIZ®v2 size

standard used for normalization

•Normalization Factor Threshold

•The passing range for Normalization Factor (default range is

0.3 to 3.0)

Normalization: Definitions

14

HID Validation:

The 3500 Series Genetic Analyzers

Sources of Variation by Normalization Method

No Norm Factory only Int Std only Factor+IntStd

Injection to Injection

Cap to Cap

Within

Instrument to Instrument

Total

39%

33%

25%

14%

31%

24%

13%

20%

19%

5%

12%

11%

4%

3%

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

Sensitivity and

GeneScan™ 600 LIZ®Size Standard v2.0

16

HID Validation:

The 3500 Series Genetic Analyzers

Sensitivity Study:

Average Peak Height across a Range of DNA Input Amounts AmpFℓSTR®

MiniFilerTM PCR Amplification Kit

Normalization Method

Factory Only

Factory + Internal Std

0 750 50 250 125

H1126

H1152

H1161 OO7

10000

5000

15000

00.125ng 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 Height

17

HID Validation:

The 3500 Series Genetic Analyzers

Sensitivity Study:

Average Peak Height by Dye for 1ng DNA Input AmpFℓSTR

®Identifiler®

PCR Amplification Kit

2000

4000

8000

10000

6000

12000

14000

Internal Standard Normalization

2000

Disabled Enabled

Peak Height

18

HID Validation:

The 3500 Series Genetic Analyzers

Size Standard Peak Height Consistency Study:

Five AmpFℓSTR®PCR Amplification Kits

GeneScan™ 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 Height

19

HID Validation:

The 3500 Series Genetic Analyzers

Size Standard Peak Height Consistency Study:

%CV for 5 Different AmpFℓSTR®Kits

GeneScan™ 600 LIZ® Size Standard v2.0

% CV

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

20

HID Validation:

The 3500 Series Genetic Analyzers

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

0Lot 1 Lot 2 Lot 3

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

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Thermo Fisher Scientific HID Validation: The 3500 Series Genetic Analyzers Owner's manual

Thermo Fisher Scientific HID Validation: The 3500 Series Genetic Analyzers Owner's manual

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