3500 Validation with

Normalization Feature

Leigh Clark, Crime Lab Analyst Supervisor, Florida Dept. of Law Enforcement – Jacksonville leighclark@fdle.state.fl.us

FDLE BACKGROUND: 6 REGIONAL FORENSIC DNA LABS 10 BIOLOGY CRIME LAB ANALYST SUPERVISORS 2 TECHNICAL LEADERS (3 LABS EACH) APPROXIMATELY 100 CRIME LAB ANALYSTS APPROXIMATELY 35 FORENSIC TECHNOLOGISTS FEW STAFF ASSISTANTS & CRIME LAB TECHNICIANS 1 QUALITY MANAGER (ALL DISCIPLINES) 1 ASSISTANT QUALITY MANAGER (ALL DISCIPLINES)

3130XLs IN USE STATEWIDE FOR > 10 YEARS WHY CHANGE?

REAL REASONS

WINDOWS – NETWORKS OTHER SOFTWARE

INCREASED THROUGHPUT INSTRUMENTS AGING

BUDGETARY

TANGENTIAL REASONS CONSISTENCY

6 DYE CAPABILITY NEW LASER & HEATER

THE ORIGINAL PLAN:

INSTALL 3500s AND SWITCH TO ID-X AND…

REALITY: 1. NEW COMPUTERS 2. SWITCH TO GMID-X V 1.4 3. 3500 INTEGRATION

3500 VALIDATION JACKSONVILLE (2)

FOLLOWED BY PERFORMANCE CHECKS

TAMPA (3) ORLANDO (3) FT MYERS (2)

PENSACOLA (2) TALLAHASSEE (2)

JACKSONVILLE

OTHER THAN BRINGING INSTRUMENTS ONLINE, WHAT DO WE HOPE TO

ACCOMPLISH? • ELIMINATE THE “FAVORITE” INSTRUMENT ISSUE

• STREAMLINE MAINTENANCE • INCORPORATE INTO CURRENT AUTOMATED WORKFLOW

• GET BACK TO OUR FULLY NETWORKED STATUS

EXPECTATIONS BASED ON INFO PROVIDED BY VENDOR?

KEEP IN MIND THIS IS A CE VALIDATION, NOT AMP KIT!

FACTORY STANDARDIZATION

NORMALIZATION?

“A method to attenuate signal variations associated with instrument, capillary array, sample salt load, and injection variability” Life Technologies, 2010

PRECISION: STUDY CONDUCTED WITH INJECTIONS OF LADDER… REPEAT INJECTIONS… DIFFERENT WELLS… DIFFERENT DAYS…

OVERALL, THE VARIATION (BP) WAS MEASURED AT 3SD TO BE WELL WITHIN THE 0.5NT RANGE NEEDED FOR CORRECT SIZING AND GENOTYPING. THE LOCUS WITH THE GREATS VARIABILITY WAS D8, WHICH STILL HAD AN EXPECTANCY OF 99.7% OF ALL ALLELES SIZE WITHIN 0.28NT… THIS INSTRUMENT WAS PRECISE!

Locus AMEL CSF D13 D16 D18 D19 D21 D2 D3 D5 D7 D8 FGA TH0 TPOX vWA Avg SD 0.04 0.06 0.05 0.06 0.06 0.05 0.06 0.07 0.05 0.06 0.06 0.09 0.06 0.06 0.06 0.06

Avg 3SD 0.12 0.18 0.14 0.18 0.18 0.14 0.17 0.21 0.15 0.18 0.18 0.28 0.19 0.17 0.18 0.17

sample run locus allele size allele size allele size allele size allele size G01_LADDER-1 043013 D13S317 8 215.17 9 219.25 10 223.27 11 227.39 12 231.41 G01_LADDER-1 043013_2 D13S317 8 215.17 9 219.25 10 223.29 11 227.34 12 231.4

size difference 0 0 0.02 0.05 0.01

REPRODUCIBILITY / CONCORDANCE: KNOWN SAMPLES GAVE THE SAME RESULTS WITH REPEAT INJECTIONS… INJECTIONS OF NIST STANDARD SAMPLES YIELDED THE EXPECTED RESULTS… SHOCKING…

SENSITIVITY / THRESHOLDS: ASSESSMENT OF BASELINE NOISE, SIGNAL TO NOISE DETERMINATION OF AN ANALYTICAL THRESHOLD DYE CHANNEL SPECIFIC APPROX 3-5X BASELINE NOISE DETERMINATION OF A STOCHASTIC THRESHOLD AT WHAT RANGE DO WE START LOSING KNOWN SISTER ALLELES?

AGAIN – THIS IS A CE VALIDATION – THE AMP KIT VALIDATION STANDS ALONE…

WAIT… WHAT ABOUT THAT NORMALIZATION FEATURE???

SO FAR, HAD BEEN USING 8.5uL OF HI-DI FORMAMIDE : 0.5uL OF GS600-LIZ

GS600-LIZ vs GS600-LIZ v2?

Basically the same product, with the same dye label and range of fragment sizes, but the v2

has been manufactured to ensure greater consistency in performance between lots.

HOW DO I DETERMINE A NORMALIZATION FACTOR?

LIZ AND LIZ V2 WITHOUT COMPETITION

LIZ AND LIZ V2 WITH AMP PRODUCT SIZE APC-K APC-Q Ladder ANC Average 200 3858 2871 2343 2911 2995.75 220 3169 2408 1937 2444 2489.5 240 4760 3640 2915 2695 3502.5 260 3911 2922 2343 3000 3044 280 4097 3131 2575 3216 3254.75 300 4311 3272 2669 3289 3385.25 314 2861 2215 1789 2153 2254.5 320 3193 2468 1988 2525 2543.5 340 4131 3046 2540 3220 3234.25 360 3493 2608 2179 2682 2740.5 400 3438 2633 2177 2617 2716.25

2923.705

THIS EXAMPLE SUPPORTS A NORMALIZATION TARGET

OF APPROX 3000 RFU

“A method to attenuate signal variations associated with instrument, capillary array, sample salt load, and injection variability” Life Technologies, 2010

In theory, the factors that affect injection quality which are beyond my control can be “evened out”

between injections by using this feature…

What do I depend on within each injection that is standardized, made available for injection at the same concentration/amount in every well? ILS

We depend on it to migrate in the same fashion with

every injection. Can it be used to normalize the injection sensitivity as well?

Is there a “sample effect” on the peak heights associated with the GS600-LIZ v2?

While I swear I have seen greater LIZ peak heights with blanks and negative controls

than with high template samples, big mixtures,

positive controls, and ladders, I simply did not observe this…

APC-K APC-Q Ladder ANC160 3846 2850 2263 2901180 4702 3625 2820 3522200 3858 2871 2343 2911214 3859 2857 2296 2814220 3169 2408 1937 2444240 4760 3640 2915 2695250 2067 1541 1272 1554260 3911 2922 2343 3000280 4097 3131 2575 3216300 4311 3272 2669 3289314 2861 2215 1789 2153320 3193 2468 1988 2525340 4131 3046 2540 3220360 3493 2608 2179 2682380 2518 1883 1593 1938400 3438 2633 2177 2617414 3287 2486 2086 2501420 2848 2167 1828 2202

If the amount of amp product doesn’t affect LIZ, then it seems like an acceptable means of normalization

The increased range of detection eliminated the need for reduced injection times for off-scale samples!

Could some of the instances where we use an increased injection time be alleviated by using the normalization feature?

Single source male at ~3.75ng target amp, 3500xl

Single source male at ~2.0ng target amp, 3130xl

Whatever impacts the injection of LIZ affects the accompanying sample…

If LIZ varies from the expectancy, it can be adjusted, and all of the other peaks adjusted by the same factor – a normalization factor.

Apply this normalization across the board, and we have basically ensured consistency within injections on the same instrument.

The 2 JROC instruments demonstrate nearly identical sensitivities out of the box, BUT, were this not the case, or if we see changes over time… normalization can help equilibrate 2 or more instruments…

LIZ V2 WITH AMP PRODUCT

FIRST INSTRUMENT AT JROC – 3214 RFU SECOND INSTRUMENT AT JROC – 3178 RFU

NORMALIZATION TARGET 3200

The 2 JROC instruments demonstrate nearly identical sensitivities out of the box, BUT, were this not the case, or if we see changes over time… normalization can help “equalize” 2 or more instruments…

3500xl #1 average LIZ peak heights = 2500 RFU 3500xl #2 average LIZ peak heights = 3500 RFU 3500xl #3 average LIZ peak heights = 4250 RFU

2500

3000

3500

4000

3500xl #1 average LIZ peak heights = 2500 RFU Allow a normalization range to at least 2.5 (2500 to 3500)

3500xl #2 average LIZ peak heights = 3500 RFU 3500xl #3 average LIZ peak heights = 4250 RFU

Allow a normalization range from at least 0.8 (4250 to 3500)

2500

3000

3500

4000

Example: 3 instruments with varying performance based on injection of identical samples…

But if the target has been determined to be 3500 RFU, why use different ranges? Let the software do the work!

Example: 3 instruments with varying performance based on injection of identical samples…

Assuming a normalization target of 3500… Using a normalization range of 0.3 to 3.0, the following occurs:

3500 Sample Avg of 11 LIZ peaks

Norm Target

Norm Factor

NF applied

1 3000 3500 1.17 1.172 1100 3500 3.18 3.001 3200 3500 1.09 1.092 3600 3500 0.97 0.971 4800 3500 0.73 0.732 5700 3500 0.61 0.61

#1

#2

#3

Whatever impacts the injection of LIZ affects the accompanying sample, whether that be sample-related or instrument related or just

by chance…

If LIZ varies from the expectancy, it can be adjusted, and all of the other peaks adjusted by the same factor – a normalization factor.

Apply this normalization across the board, and we have basically ensured consistency

between instruments.

Could some of the instances where we use an increased injection time be alleviated by using the normalization feature?

Norm Target

Min Norm Threshold

Max Norm Threshold

On the first instrument assessed, the average height of LIZ peaks across the 11 fragments of interest for >80 injections of all sample types was 3214. NT = 3200 was selected for remaining sensitivity and mixture studies.

9947A 0.50ng target, 3130xl

For good quality data, life doesn’t change much other than the Y axis and dynamic range…

9947A 0.50ng target, 3500, not normalized

9947A 0.50ng target, 3500, normalized (NF=1.362)

Note this is a separate injection

Typical rare sperm fraction – can see some alleles below threshold… increase injection time? Did something affect the entire injection? How does the LIZ look for this sample?

Same typical rare sperm fraction – normalization factor 1.86 (LIZ peaks for this sample were only around 1700 RFU – normalization was warranted)

At first, a norm range of 0.5 to 2.0 was selected. In initial experiments, any samples that required

normalization > 2.0 were “problem samples”.

Normalizing up was well-accepted.

Any risks associated with normalizing down?

With good target amp samples, no issues observed.

What about low level samples? (again, the new CE platform has no effect on what

already happened in the amp…)

Sample ID 8:4:1 8:4:1 8:4:1 8:4:1

Description 1.0ng 1.0ng 1.5ng 1.5ng

Normalization no NF NF=0.813 no NF NF=0.678

D8S1179 11,14,15 11,14,15 11,14,15 11,14,15

D21S11 29,30,30.2,31,32.2 29,30,30.2,31,32.2 29,30,30.2,31,32.2 29,30,30.2,31,32.2

D7S820 7,9,10,11 7,9,10,11 7,9,10,11 7,9,10,11

CSF1PO 11,12,14 11,12,14 11,12,14 11,12,14

D3S1358 15,16,17 15,16,17 15,16,17 15,16,17

TH01 7,9.3 7,9.3 7,9.3 7,9.3

D13S317 11,12,13 11,12,13 11,12,13 11,12,13

D16S539 9,11,12,13,14 9,11,12,13,14 9,11,12,13,14 9,11,12,13,14

D2S1338 16,18,20,22 16,18,20,22 16,18,19,20,22,25 16,18,19,20,22,25

D19S433 12,13,14 12,13,14 12,13,14,14.2 12,13,14,14.2

vWA 14,16,17 14,16,17 14,16,17 14,16,17

TPOX 8,11,12 8,11,12 8,11,12 8,11,12

D18S51 13,15,16 13,15,16 13,15,16 13,15,16

AMEL x,y x,y x,y x,y

D5S818 11,12,13 11,12,13 11,12,13 11,12,13

FGA 22,23,25,26 22,23,25,26 22,23,25,26 22,23,25,26

Sample ID 8:4:1 8:4:1

Description 0.5ng 0.5ng

Normalization no NF NF=0.8

D8S1179 11,14,15 11,14,15

D21S11 30,30.2,31,32.2 30,30.2,31,32.2

D7S820 9,10,11 9,10,11

CSF1PO 11,12,14 11,12,14

D3S1358 15,17 15,17

TH01 7,9.3 7,9.3

D13S317 11,12,13 11,12,13

D16S539 9,11,12,14 9,11,12,14

D2S1338 16,18,20,22 16,18,20,22

D19S433 12,13,14 12,13,14

vWA 14,16,17 14,16,17

TPOX 8,11,12 8,11,12

D18S51 13,14,15,16 13,14,15,16

AMEL x,y x,y

D5S818 11,12,13 11,12,13

FGA 22,23,25,26 22,25,26

Is normalization worth the loss of a single called allele? Note the NF here is 0.8, so the LIZ peaks in this sample averaged 4000 RFU versus the target of 3200 RFU. How would you know about this possible additional allele?

Repeat injections of same sample where NF = No Same sample in a different well where NF = 1.243 and 1.134 for repeat injections.

Normalization Factor

Locus Allele RFU Allele RFU Allele RFU Allele RFU Locus Allele RFU Allele RFUD8 12 330 13 263 12 504 13 378 D8 12 131 13 103

D21 28 117 31 141 28 184 31 223 D21 28 56 31 68D7 7 12 7 165 12 192 D7 7 12CSF 12 410 12 589 CSF 12 166D3 15 393 16 419 15 576 16 646 D3 15 144 16 149

TH01 9.3 209 9.3 296 TH01 9.3 83D13 11 794 11 1196 D13 11 335D16 9 10 265 9 158 10 380 D16 9 52 10 113D2 20 172 23 20 264 23 D2 20 86 23

D19 14 15 14 15 123 D19 14 15vWA 14 205 16 135 14 302 16 191 vWA 14 82 16TPOX 8 540 8 886 TPOX 8 201D18 12 347 15 297 12 533 15 470 D18 12 136 15 105

Amel x 145 y x 282 y Amel x 60 yD5 11 291 11 406 D5 11 129

FGA 24 26 163 24 125 26 259 FGA 24 26 63

3500xlLow template 007 run on 3130xl and 3500xl with and without normalization

3130xl

<STOdrop-out3130xl AT=50 STO=200; 3500xl AT=100; STO=350

Not Normalized NF = 1.528

Locus 3500xl3500xl

NF=1.5283130xl KNOWN

D8 12,13 12,13 12,13 12,13D21 28,31 28,31 28,31 28,31D7 NSD 7,12 NSD 7,12CSF 12,12 12,12 12+ 12,12D3 15,16 15,16 15,16 15,16

TH01 9.3+ 9.3+ 9.3+ 9.3,9.3D13 11,11 11,11 11,11 11,11D16 10+ 9,10 9,10 9,10D2 20+ 20+ 20+ 20,23D19 NSD 15+ NSD 14,15

vWA 14,16 14,16 14+ 14,16TPOX 8,8 8,8 8,8 8,8D18 12,15 12,15 12,15 12,15

Amel x+ x+ x+ X,YD5 11+ 11,11 11+ 11,11

FGA 26+ 24,26 26+ 24,26

Example of the benefits of up-normalization on a single instrument: • 3130xl results would likely have prompted an increased injection • 3500xl (not norm) results very similar • Normalization allowed for 2 additional GT calls & 1 additional

single allele call…

Run Module

Assays contain: Instrument protocol, Primary analysis protocol (QC for HID), Optional secondary analysis protocol (ID-X for HID)

Run Module → Instrument Protocol + Primary Analysis

Protocol → Assay

Assay applied to plate record; primary analysis at collection, norm applied

MANY THANKS! Applied Biosystems

Life Technologies Thermo Fisher Scientific Specifically Melissa Kotkin & Shelly Guerrero

Florida Department of Law Enforcement

JROC Chief of Forensics Lisa Zeller

Leigh Clark, Crime Lab Analyst Supervisor, Florida Dept. of Law Enforcement – Jacksonville leighclark@fdle.state.fl.us