Outline Scope Materials and Reagents Validation Elements Selection of Validation Data Additional Sources of Validation Information User Bulletin 2
Validation Scope Hardware and Reagents HID Validation: 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 3
Validation Scope Sample Summary HID Validation: 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 Minifiler: 0.125ng, 0.25ng, 0.5ng,.75ng 5 replicates for DNA samples 3 replicates for NTC 4 injections 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 4
Validation Scope AmpFlSTR Kits Identifiler Minifiler SGM Plus Yfiler Sinofiler SEfiler Plus Profiler Plus Cofiler Identifiler Direct Identifiler Plus NGM 5
Precision and Accuracy
Precision and Accuracy Example Configurations for Standard Deviation Within Injection - 3500 Across Injections 3500xL 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 7
3500 Sizing Precision Study: Within an Injection of Identifiler Allelic Ladder 8
3500 Sizing Precision Study: Across Multiple Injections of Identifiler Allelic Ladder Samples 3500-12 Injections 3500xL - 4 Injections 96 Replicates of Identifiler Allelic Ladder 9
Introduction to Normalization
Sources of CE Variation (3100) Raman Intensity, 3100 file: INSTRUMENT DATA Mod2B.xls Numerous optical components, capillary array 9000 8000 7000 y = -2.7202x + 6042.8 R 2 = 0.0059 Electrophoresis, injection Sample and sample preparation A/D Units 6000 5000 4000 3000 2000 median= 6026.50 Stdev = 808.54 Average = 6054.11 Stdev/ average = 0.13 Series1 Linear (Series1) 1000 0 0 20 40 60 80 100 120 140 Instrument Almost 2-fold difference in signal from over 100 instruments from the factory floor 11
Normalization: What is it? HID Validation: 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 12
Normalization: Definitions HID Validation: 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) 13
Sources of Variation by Normalization Method 39% Instrument to Instrument largest source of variation 14 %CV Internal standard Normalization greatest reduction in variation 40% 35% 30% 25% 20% 15% 10% 5% 0% 33% 31% 25% 24% 24% 20% 19% 12% 11% 5% 4% 3% 3% 0% No Norm Factory only Int Std only Levels of Normalization 0% Factor+IntStd 4% 14% 13% Total 5% Instrument to Instrument Within Cap to Cap Injection to Injection Sources of Variation
Sensitivity and GeneScan 600 LIZ Size Standard v2.0
Sensitivity Study: Average Peak Height across a Range of DNA Input Amounts AmpFlSTR MiniFiler TM PCR Amplification Kit H1126 Genomic DNA #1 0.125ng 0.25ng 0.50ng 0.75ng H1152 Genomic DNA #2 15000 10000 5000 H1161 Genomic DNA #3 OO7 Control DNA Standard 007 0 Peak Height 15000 10000 5000 0 00.125ng 00.25ng 0.50ng0 5 0.75ng Normalization Method Factory Only Factory + Internal Std 16
Sensitivity Study: Average Peak Height by Dye for 1ng DNA Input AmpF lstr Identifiler PCR Amplification Kit 14000 12000 Peak Height 10000 8000 6000 4000 2000 2000 2000 17 Disabled Enabled Internal Standard Normalization
Size Standard Peak Height Consistency Study: Five AmpFlSTR PCR Amplification Kits GeneScan 600 LIZ Size Standard v2.0 7000 6000 Peak Height 5000 4000 3000 2000 GS600 Peak Height Identifiler Identifiler Direct Identifiler Plus Minifiler TM NGM TM 1000 0 Lot 1 Lot 2 Lot 3 GS600 Lot Number 18
Size Standard Peak Height Consistency Study: %CV for 5 Different AmpFlSTR Kits GeneScan 600 LIZ Size Standard v2.0 % CV 19 Size Standard Peak Height Consistency 6 instruments 3 lots of size standard
Internal Standard Normalization: Injection to Injection Peak Height Consistency GeneScan 600 LIZ Size Standard v2.0 40 30 Normalization Method Factory Only Factory + Internal Std % CV 20 10 0 Lot 1 Lot 2 Lot 3 Size Standard Peak Height Consistency 6 instruments 3 lots of size standard 20
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) 22
Baseline Noise - Developmental Evaluation G5 Average Noise (10x SD) 10x Average Std. Dev. 200 180 160 140 120 100 80 60 40 175 RFU 20 0 FAM VIC NED PET LIZ TOTAL Data generated during the Sensitivity experiment was then used to confirm the level of pull up and the peak amplitude threshold setting. 23
Pull-up 360 Samples at 6 ng template HID Validation: 24000 20000 16000 12000 8000 4000 5.0% 24
Additional Studies and Validation Documentation
Identifiler Direct HID Validation: 14000 8000 24 Capillary 3500xL 0 8 Capillary 3500 14000 8000 0 82 gdna, 25 cycles Three 3500xl and three 3500 instruments. Mother plate split into 6 daughter plates. 26
Additional Studies HID Validation: 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 27
Validation Documentation HID Validation: AAFS and ISHI Posters Bode Conference Presentations User Bulletin containing developmental validation in progress www.appliedbiosystems.com/3500hid 28
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