Jarrah R. Kennedy

Kansas City Police Crime Laboratory

The information, data and opinions in the following presentation are those of this author and the DNA unit at the Kansas City Police Department Crime Laboratory.

As always, the parameters, procedures and limitations regarding the internal validation of quantitation and amplification chemistries along with genetic analyzers are unique to each testing laboratory.

Finally, Life Technologies does not necessarily endorse the conclusions in this presentation.

Kansas City Police Crime Laboratory – KC, MO DNA Supervisor + 7 qualified DNA analysts (includes TL) 1 analyst in training who is technically qualified in all lab work 2 additional analysts that screen and extract/amp references Extraction format:

o (2) EZ1 for all casework/unknown samples and differentials o ForensicGEM reagents for reference extraction – use heat block/96 well

Separate workflows for unknowns and references o References are not quantitated o Expert system analysis of references (10% global filter) o Utilize “batch” reviews for both workflows (lot #s, controls etc)

(1) 3130 & (1) 3500 Hamilton Starlet ID – will be validated…. Soon! (quant/amp)

3500 Genetic Analyzer: Precision/Reproducibility; Analytical Threshold (GF) Standard assay conditions (run voltage, injection time) for GF on 3500 Analysis parameters with GMIDX v1.4 Correlation of Trio sensitivity/stochastic study with GF sensitivity Stability of Trio standards over time and storage (tube type) GF Sensitivity* and Stochastic threshold on 3500

o Samples A & B : 5ng – 15.625ng (12 samples in triplicate) o Sample C examined stochastic range: 230 pg – 7.8 pg (6 samples in triplicate)

Mixtures - correlation of Trio ratio with GF observed ratio o 2-person male:female mixtures in 50:1 to 1:1 (11 samples in duplicate) o Complex 3,4 and 5 person mixtures in varying ratios (at 1 and 2/2.5ng)

Inhibition – correlation of Trio IPC/DI to GF results o Humic Acid (70, 140 and 210 ng/ul) o EDTA reconcentration

Degradation – correlation of Trio DI to GF results o Heat and humidity for different times

Non-probative/mock casework samples Several other mini-studies evaluating:

o Expectations of altered injection times o Polymer Life on the 3500 7 14 days o Injection sample prep stability

Trio specific results

Slope: -3.0 to -3.7 o Visual examination & adjustment for parallel slopes o Non-similar slopes will affect DI and M:F Ratio

R2: ≤ 0.98 IPC for standards: 25.5 to 30.5 IPC for samples: variance ≥ 1.5

o The IPC average calculated per run – cannot input a calculated average

Low Quant: ≤ 0.0050 o May not utilize this flag – doesn’t work for undetermined value

Standard Curve Evaluation Results

Standards: Full Volume

(n = 6)

Standards: Half Volume

(n = 6)

Aged* Standards: Full Volume

(n = 6)

%CV

Large Autosomal (LA target)

Slope -3.502 -3.505 -3.510 3.3%

Y-intercept 24.964 24.845 24.946 0.9%

R2 0.999 0.997 0.998 0.2%

Small Autosomal (SA target)

Slope -3.340 -3.377 -3.411 3.1%

Y-intercept 27.252 27.199 27.429 0.9%

R2 0.999 0.998 0.998 0.2%

Y target

Slope -3.393 -3.439 -3.420 3.3%

Y-intercept 26.319 26.264 26.477 0.8%

R2 0.999 0.996 0.998 0.3%

R2 > 0.992 for all curves *25 days old

Slope Average %CV Ave %CV HIGH LOW DIFF

Full volume (n = 6)

LA -3.502 3.7%

3.4%

-3.396 -3.739 0.34

SA -3.340 3.2% -3.235 -3.51 0.28

Y -3.393 3.4% -3.295 -3.604 0.31

1/2 volume (n = 6)

LA -3.505 4.0%

3.4%

-3.326 -3.64 0.31

SA -3.377 2.6% -3.239 -3.464 0.23

Y -3.439 3.5% -3.256 -3.542 0.29

Y-Intercept Average %CV Ave %CV HIGH LOW DIFF

Full volume (n = 6)

LA 24.964 0.8%

0.8%

25.224 24.711 0.51

SA 27.252 0.8% 27.486 26.959 0.53

Y 26.319 0.7% 26.549 26.082 0.47

1/2 volume (n = 6)

LA 24.845 1.3%

1.1%

25.392 24.456 0.94

SA 27.199 1.1% 27.685 26.775 0.91

Y 26.264 0.8% 26.487 25.977 0.51

6 different curves prepared over 6 runs with 5 different analysts There are NO OBSERVABLE DIFFERENCES: -½ or full volume -age of standards -Tube type for standards Lot to lot variability is possible; this may be due to stochastic sampling (1ul in ½ volume) 1 Ct shift 2-fold quant diff

If slopes are not similar to each other, mixture ratio and degradation index will not be accurate

5 analysts, 2 volumes (full/half), 3 curves per volume = 30 standard curves

11 out of 30 had to be adjusted because of slope o Usually omit Standard D or E o 3 Large Autosomal o 3 Small Autosomal o 5 Y o Can omit 1 of the 3 – or the whole standard

Two different lots of Trio examined o 1312001 - first released lot o 1403002 – only one run performed with this lot

• Will perform more quant runs through implementation training

½ volume reaction appears slightly more accurate at higher concentrations of the standard curve as well as the undiluted Trio standard – but overall each is similar in difference.

Expected (ng/ul)

Full Volume (ng/ul) % Diff 1/2 Volume

(ng/ul) % Diff

NIST2372-A 57 50.4 -11.5% 56.6 -0.6%

NIST2372-B 61 48.6 -20.4% 55.9 -8.3%

NIST2372-C 59 67.2 13.8% 71.0 20.4%

NISTstd A 50 46.9 -6.3% 55.7 11.4%

NISTstd B 5 5.78 15.6% 7.34 46.8%

NISTstd C 0.5 0.58 15.5% 0.76 51.9%

NISTstd D 0.05 0.045 -9.9% 0.055 9.8%

NISTstd E 0.005 0.003 -37.7% 0.005 10.0%

Stock Trio Standard 100 82.9 -17.1% 109.3 9.3%

Average 16.4% 18.7%

Dilution series from 4 individuals made (ng/µl) o 66.5, 38, 5.43, 0.776, 0.111, 0.016, 0.0023, 0.00032, 0.000046

Drop-out starts at 0.00032 ng/µl (0.32pg/µl) o 0.00032 ng/µl x 15 µl = 4.8pg DNA

Conc. (ng/ul)

Full Volume (# replicates w/value)

Half Volume (# replicates w/value)

Larg

e Au

toso

mal

0.0023 8/8 8/8

0.00032 8/8 6/8

0.000046 2/8 1/8

Smal

l Au

toso

mal

0.0023 8/8 8/8

0.00032 8/8 8/8

0.000046 1/8 2/8

Y

0.0023 4/4 4/4

0.00032 3/4 3/4

0.000046 1/4 0/4

14-010

Average DNA Concentration

(ng/µl) % Difference ½ Volume

full volume n=10

½ volume

n=8

FULL vs. ½ VOLUME

SA vs Male (½ volume)

Highest Observed

(ng/µl)

Lowest Observed

(ng/µl) %CV

Human (small)

5.125 5.089

-0.7% -7.7%

5.934 3.861 13.1%

Male 4.840 4.698 5.168 3.846 8.4%

14-010

Average Ct Value %CV

full volume n=10

½ volume n=8

full volume n=10

½ volume n=8

Large 22.94 22.85 0.4% 0.8%

Small 24.91 24.77 0.4% 1.4%

Y 24.03 23.98 0.3% 0.8%

Consistency of a value with repeated examination (Trio). 14-010 will be the internal QC control for Trio quantitation kits and is a male standard. Similar results as with standard curves; no observable differences between analysts, reaction volume, tubes or age of standards. Again, ½ volume has slightly more variance, but still a low level of variance observed.

Based upon the similarity in the quantitation values for full and ½ volume quantitation reactions- the KCPCL has decided to proceed with ½ volume reactions.

However, we will monitor the y-intercept values of the ½ volume reaction with training samples and possible lot variation. o 1 Ct difference 2-fold difference in quantitation value

½ volume sensitivity at low end is reliable – 0 means 0

½ volume quantitation of concentrated samples may be slightly

more variable; however due to robust range of GF/3500 – not really concerned (plenty of DNA, right? Re-amp? Reduce injection time?)

Degraded & Inhibited Samples

IPC flag set to fire at 1.5 Ct > IPC average for that quant run o IPC flag can fire with a highly concentrated sample, but typically won’t fire if

concentration ≤ 50 ng/µl Degradation Index (per user guide and previously discussed!)

o <1 = no degradation present o 1-10 = some degradation or inhibition is present o >10 = sample is significantly degraded or inhibited

½ volume had 20% higher DI than full volume o Possibly a better indicator with ½ volume? The degradation observed may

have been more extensive than scale above (what is significant to you?!) Stochastic samples can demonstrate elevated DI due to poorer

amplification of the LA target in these samples Humic Acid & EDTA reconcentrated samples from 200 and 400µl

of TE (versus water reconcentration controls)

Humic Acid Concentration

(ng/ul)

Trio IPC results

GlobalFiler # alleles >175rfu / total alleles

KCPD Applied Biosystems KCPD Applied

Biosystems

70 Normal (27.19) NA 46/46 43/43

140 Normal (27.74) Normal 46/46

(lower PH) 43/43

210 Elevated (30.71) NA 20/46 39/43

300 NA Elevated NA NA

Sample Deg Index IPC Ct ng DNA PH/A Comments

70 ng/ul Humic Acid 1.4 27.19 1.26 5771.0 No quality issues observed

140 ng/ul Humic Acid 5.2 27.74 1.54 2410.9 Full profile, but lower PH – see profile

210 ng/ul Humic Acid 30.71 1.33 4623.6 No LA quant; 20/46 alleles – see profile

Control_TE_A 0.6 27.36 1.45 10964.2

Control_H2O_A 0.8 27.19 1.35 13686.6

200ul_TE_A 0.9 27.18 0.40 2687.0 2 loci < 60% PHR

200ul_H2O_A 0.8 27.18 1.58 5854.3

400ul_TE_A 1.1 27.16 1.60 2156.7 1 locus <60% PHR

400ul_H2O_A 0.7 27.14 1.42 5940.8

Control_TE_B 0.7 27.12 1.44 10610.0

Control_H2O_B 0.8 26.94 1.10 9552.3

200ul_TE_B 0.7 27.10 1.08 5818.4 Split peaks; 37/46 alleles – see profile

200ul_H2O_B 0.7 26.99 1.00 12486.3

400ul_TE_B 0.7 27.05 1.17 2535.1 Split peaks; 32/46 alleles – see profile

400ul_H2O_B 0.8 26.96 1.00 13943.4

IPC = 27.74 DI = 5.2 Also lower at D7, D13 (not pictured)

IPC = 30.71 DI = NA (no LA quant)

IPC = 27.11 DI = 0.7 IPC = 26.99

DI = 0.7 TE

H2O

IPC = 27.05 DI = 0.7

IPC = 26.96 DI = 0.8

TE

H2O

EDTA inhibition did not cause an increase in the DI or the IPC – but you saw it in the profiles?

Per Life Tech; this is due to more MgCl2 present in the Trio

reaction (this excess binds the EDTA) than the GF reaction. GlobalFiler cannot have as much MgCl2 due to multiplexing – so GF will be more sensitive elevated EDTA than Trio.

Important for us due to workflow for STR/Y-STR analysis and

possible reconcentration of samples eluted in TE for Y amplification. **we have updated our SOP – water elution if reconcentration will occur**

As expected; inverse Relationship between Degradation Index and the % of alleles detected. Lower the DI – the more alleles detected for degraded samples.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Perc

enta

ge o

f Alle

les

Det

ecte

d

Degradation Index

Correlation of Degradation Index to DNA Profile Obtained

Full Volume

Half Volume

GlobalFiler

Ladder Precision/Reproducibility SD = 0.04 bp (<< 0.15bp) No carryover observed unless overblown injection and

subsequent 30 second injection (not-normal injection conditions or samples)

Standard Instrument Protocol/Assay (5-30sec validated)

11kV run voltage was examined to evaluate resolution of single base pair differences

11kV (201 RFU) only gained 1

peak vs. 13kV. The most significant gains in base pair resolution were through the peak window size reduction from 13 11

Analytical Threshold o Analyzed duplicate injections of triplicate amplifications of 2 different dilution series ranging from 5ng to

15.63pg of DNA o Highest anomalous peak +3D 175 RFU o Utilized 3 sensitivity series (A,B & C) plus mixture and other empirical data.

Stochastic Threshold

AT*[1/(Ave PHR – 3*SD-PHR)]

o Evaluating 600 – 800 RFU (likely will be 700 RFU) Average difference in peak heights between duplicate injections of same sample = 4.5%. Can be as

high as 19%. Polymer life verified for 2 full weeks Removal of denaturation/snap-cool step verified Sample prep life for 4 days (some decrease in PH observed – not outside of expected variation)

Recommended Stutter A Observed + 3SD B Observed + 3SD

Marker - st % + st% - st % + st% - st % + st% D3S1358 10.98% 7.77% 8.71% vWA 10.73% 6.41% 7.40% D16S539 9.48% 6.15% 7.56% CSF1PO 8.77% 5.97% 6.85% TPOX 5.55% 3.20% 3.25% D8S1179 9.60% 6.91% 7.96% D21S11 10.45% 6.68% 7.68% D18S51 12.42% 7.67% 6.63% DYS391 7.43% 5.54% 6.36% D2S441 8.10% 4.19% 3.57% D19S433 9.97% 7.99% 8.03% TH01 4.45% 2.24% 3.28% FGA 11.55% 7.03% 7.51% D22S1045 (trinuct) 16.26% 6.69% 9.42% 5.15% 8.10% 3.70% D5S818 9.16% 6.61% 6.63% D13S317 9.19% 3.21% 5.91% D7S820 8.32% 2.78% 5.36% SE33 (-2) 3.97% 3.68% 3.92% SE33 (-4) 14.49% 9.70% 11.32% D10S1248 11.46% 9.46% 8.38% D1S1656 (-2) 2.45% NA NA D1S1656 (-4) 12.21% 7.29% 10.64% D12S391 13.66% 6.37% 8.42% D2S1338 11.73% 8.65% 9.79%

Trinucleotide locus: D22S1045 - +3 stutter observed -2 stutter observed at: SE33 D1S1656 Went with recommended settings as observed + 3SD did not exceed the stutter panel values

Sensitivity A & B: Dropout begins at 250 pg (not in all reps)

500pg corresponds to where average PH/A < 1000 RFU o Increased variability in PHR and PH across the sample begin here

Sensitivity series C – more sensitive sample (7.8pg = 0.52pg/ul quant)

SENSITIVITY - SAMPLE A - STUDY #1 Alleles Detected > 175 RFU

Sample 1 2 3 4 5 6 0.25 43/43 43/43 42/43 42/43 43/43 43/43

0.125 33/43 30/43 36/43 31/43 36/43 34/43 0.0625 10/43 6/43 7/43 7/43 12/43 12/43

0.03125 4/43 3/43 5/43 6/43 4/43 4/43

0.01563 1/43 1/43 0/43 0/43 0/43 0/43

SENSITIVITY - SAMPLE B - STUDY #1 Alleles Detected > 175 RFU

Sample 1 2 3 4 5 6 0.25 44/44 43/44 44/44 44/44 44/44 44/44

0.125 33/44 34/44 30/44 28/44 43/44 43/44 0.0625 13/44 12/44 4/44 4/44 19/44 20/44

0.03125 4/44 4/44 2/44 1/44 5/44 7/44

0.01563 1/44 1/44 0/44 0/44 0/44 0/44

SENSITIVITY - SAMPLE C (Study #2) Alleles Detected > 175 RFU

Sample Rep 1 Rep 2 Rep 3 230pg 46/46 46/46 46/46

117pg 46/46 46/46 46/46 62.5pg 46/46 46/46 46/46

31.25pg 43/46 45/46 42/46

15.625pg 28/46 38/46 26/46

7.8pg 14/46 13/46 22/46

DNA

D8

D21

D7

CSF

D3

TH01

D13

D16

D2(S1)

D19

vWA

TPOX

D18

Amel

D5

FGA

D1

D10

D12

D2(S4)

D22

DY3

SE33

Yind

Average# alleles obtained / total

alleles

500pg

43/43

250pg

42/43

125pg

33/43

62.5pg

10/43

31.25pg

4/43

15.63pg

1/43

0.125 ng

31.25pg

62.5pg

973 RFU ~300 RFU

190 RFU

356 RFU

285 RFU

62.5pg

0.125 ng

31.25pg

850 RFU

320 RFU

Optimal Template Input – Empirical & Sensitivity A-B

Red circle indicates target for PH/A – below black line (7500RFU)

should prevent most spectral peaks from affecting interpretation if spectral corrects 1% - 5% [175/7500 – 2.3%]

Increased Injection Time Sample 15sec (PH/A) 30sec (PH/A) Factor Increase

230pg 2653.1 5639.6 2.1

117pg 1091.7 2344.8 2.1

62.5pg 912.2 2136.1 2.3

31.25pg 465.9 938.6 2.0

15.625pg 322.9 608.5 1.9

7.8pg 253.4 401.9 1.6

Decreased Injection Time % 10sec of 15sec Increase Factor

Sample ng Day 1 Day 4 Day 1 Day 4

KCPD_NIST 1.5 64.2% 70.6% 1.6 1.4

NIST A 1.5 67.4% 58.7% 1.5 1.7

NIST C 2 65.4% 62.7% 1.5 1.6

NIST E 1.5 65.4% 59.7% 1.5 1.7

NIST F 1.5 65.1% 66.4% 1.5 1.5

NIST D 1.91 67.0% 60.5% 1.5 1.7

Average 65.8% 63.1% 1.5 1.6

**Predictable and proportional increases or decreases with the increase or decrease in injection time. **No differences observed based upon locus size (which we see in our lab with 3130). **No change in instrument noise (AT) with elevated injection time

15 Sec

30 Sec

220

496

279

640

Known mixture ratios prepared, quantitated and amplified: 1:50;1:20; 1:10;1:5;1:2;1:1;2:1;5:1;10:1;20:1;50:1

Concordance between Trio ratio and the GlobalFiler calculated Major:Minor ratio

Dropout of the minor component began at 1:20 prepared dilutions (Trio ratio of 12:1)

Detection of the minor component at the 1:50 prepared dilutions (Trio ratio of 36:1)

Increased template amplification potential at least double peak heights (observe 1-9 factor increase) and gain 16-50% of the minor component, depending on the position of the minor (stutter, spectral) and the observed ratio.

Mixtures (T1311)

Average Ratio Obtained

Increase Template –Mixtures Comparison by TPH

Expected Trio GlobalFiler

1ng [T1311]

2 or 2.5ng

[T1318]

Factor Increase

Minor Gain

Mix

ture

Set

A

50:1 36.74 36.28

1:50-A1 623561.5 706046 1.1 0/36 20:1 12.13 15.65

10:1-A1 310479.0 413753 1.3 Full at 1ng

10:1 5.64 7.19

50:1-A1 303069.5 516917 1.7 12/38 5:1 2.85 3.84

1:50-B2 322045.0 583090 1.8 10/33

2:1 NA 1.52

50:1-B2 251258.0 526428 2.1 8/35 1:1 <1 0.80

1:50B2- 30sec 675411.0 NA 2.7 7/33

0.50 (1:2) 0.40

1:50-B1 258773.5 557749 2.2 10/33 0.20 (1:5) 0.19

50:1-B1 257188.0 582846 2.3 10/35

0.10 (1:10) 0.10

1:1-B1 287306.5 718670 2.5 Full at 1ng 0.05 (1:20) 0.06

1:20-A2 277280.0 712488 2.6 6/36

0.02 (1:50) 0.04

5:1-B2 302881.0 937552 3.1 Full at 1ng

Mix

ture

Set

B

50:1 36.16 27.85

20:1-B1 95080.5 388267 4.1 16/35 20:1 15.92 18.18

1:20-A1 179122.0 799801 4.5 14/36

10:1 9.53 10.95

1:2-A1 106745.5 493660 4.6 1/36 5:1 5.14 5.43

2:1-A1 111618.0 629700 5.6 Full at 1ng

2:1 1.88 2.84

20:1-B2 61936.0 572418 9.2 11/35 1:1 1.06 1.12

1:50-A2 (LPH) NA 781000 NA 12/36

0.50 (1:2) 0.61

10:1-A2 (LPH) NA 558217 NA Full at 2ng 0.20 (1:5) 0.25

5:1-B1 (LPH) NA 760698 NA Full at 2ng

0.10 (1:10) 0.13

50:1-A2 (LPH) NA 340248 NA 13/38 0.05 (1:20) 0.14

0.02 (1:50) 0.04

Increased Injection Time - Mixtures (T1409)

Sample 15 sec

30 sec

Factor Increase

1-10A_2 2/36 13/36 2.1

1-10B_1 FULL FULL 2.0

1-20A_1 18/36 24/36 2.2

1-20B_1 13/33 19/33 2.0

1-20B_2 11/33 20/33 1.9

1-2A_1 35/36 35/36 2.1

1-5A_2 17/36 28/36 2.0

20-1A_1 32/38 35/38 2.0

20-1B_1 7/35 14/35 2.5

5-1B_1 9/35 21/35 2.1

50-1A_1 10/38 20/38 2.2

50-1A_2 1/38 6/38 2.1

Average Factor Increase: 2.1

Mixtures were also subjected to increased injection times to attempt to detect more minor component. The factor increase and the gain in obligate minor alleles are listed.

17 total: (8) 3-person; (6) 4-person and (3) 5-person Trio quantitation/expected and GF ratio are consistent 2ng template > double PH (typically) and gain alleles Two majors discernible at ≥ 4:4:1 Major contributor is clear at ≥ 8:1:1, 8:1:1:1, 8:1:1:1:1 4:1:1 may be partially discernible (or for CODIS) Some other mixtures may be suitable for forensic mixtures Only one 5 person mixture demonstrated > 8 alleles (D1S1656 locus) to

indicate at least 5 contributors – masking should still be expected Most common polymorphic loci for max alleles observed:

o D1S1656 (9/17) o SE33 (7/17) o D12S391 (5/17)

Ave Ratio Obtained GlobalFiler

Description Expected Trio Alleles Locus Description Ratio

3 Pe

rson

CM1-3 4M:4M:1M 1 6 D18/D1 2 majors (1:1) and 1 minor in progress

CM2-3 2F:1M:1M 1 1.15 6 SE33/D12S391 at least 3 contributors

CM3-3 4M:1F:1M 1 6 SE33/D21338 at least 3 contributors

CM4-3 8F:1M:1F 9 9.43 6 D1S1656/D12S391* 1 major w/ at least 2 minor 10 (3.7-16)

CM5-3 10M:1F:1F 1 6 D1* 1 major w/ at least 2 minor 12.7 (5-35)

CM6-3 1F:1M:1M 1 5 vWA/TH01/SE33/D1 at least 3 contributors

CM7-3 8M:8F:1M 1 6 D12S391 2 majors (1:1) and 1 minor 1.2 (0.8-2.7)

CM8-3 2M:2F:1M 1 6 D1S1656 at least 3 contributors

4 Pe

rson

CM1-4 4M:4M:1M:1M 1 8 D1S1656 at least 4 contributors

CM2-4 2F:1M:1M:1M 1 6 SE33/D1/D12 at least 3 contributors

CM3-4 4M:1F:1M:1M 1 7 SE33/D1S1656 at least 4 contributors

CM4-4 8F:1M:1F:1M 4.5 4.48 6 SE33 1 major w/ at least 2 minor 6 (3.7 - 16.3)** CM5-4 10M:1F:1F:1M 1 7 D2S1338 at least 4 contributors

CM6-4 1F:1M:1M:1M 1 7 D1 at least 4 contributors

5 Pe

rson

CM1-5 4M:4M:1M:1M:1F 1 9 D1* at least 5 contributors

CM3-5 4M:1F:1M:1M:1F 1 8 D2S1338/SE33 at least 4 contributors

CM4-5 8F:1M:1F:1M:1F 5 4.96 7 D13/D12 1 major w/ at least 3 minor 8 (2.9 - 16)*** *at 2ng template only **most ratios arund 5 ***average of D13/D12 = 4.7

Expected results with some challenged samples Several hairs evaluated ; 67pg no profile at 15 second

standard injection (indications below threshold; 30 second injection attempted with 1 allele detected)

Will definitely stop at “0” quant o We currently do this due to extensive internal validation (Duo-ID)

May consider stopping ~50pg DNA – always sample dependent

Will run several more NP’s through internal training and competencies for analysts

PHR Guidelines (Single Source Profiles)

Template Observed PH Expected PHR

>500 pg >1000 RFU 60%

250-500 pg 500-1000 RFU 50%

62.5-125 pg 175-500 RFU <40%

Mixture Interpretation PHR Guidelines

Observed PH Expected PHR - Minor

>1000 RFU 50%

500-1000 RFU 40%

175-500 RFU <40%

These values may change as we proceed through additional training samples and more non-probative casework. We will stop at “0” quants. May stop

AT– 75 RFU ; ST will likely be 300 RFU o Same ratio as 3500: 175 RFU / 700 RFU

Sensitivity A – all alleles detected at 250pg, but PHR < 60% observed first at this template

1.5ng target for 3500 matches up perfectly with 3130 sensitivity (Average PH/A ~3000 RFU)

5620

4005

2923

1836 1496

893

461 231 143 110 90 97 0

1000

2000

3000

4000

5000

6000

Average Peak Height Per Allele - Series A

1 2 30.25ng 43/43 43/43 43/430.125ng 33/43 37/43 33/430.625ng 10/43 10/43 14/430.3125ng 3/43 4/43 3/430.015625ng 1/43 0/43 0/43

ng DNA# alleles obtained /total

3130:

3500 Direct Comparison:

All data is currently in review by TL o Trio training established and internal training has occurred

FINISH 3130-GF Data Analysis (Sensitivity B, Mixtures, etc)

GlobalFiler ½ volume amplification of standards (no quantitation)

Verification of YFiler utilizing Trio quantitation (sensitivity and

target determination)

GMIDX v1.4 verification (all data analysis was done with temp copy of v1.4)

Expert system workflow (have 700 references extracted; we’ve done it once, we can do it again!)

Quantifiler® Trio Validation Lead – Jennifer McMurray GlobalFiler™ and 3500 GA Validation Lead–Jarrah Kennedy GlobalFiler™ ½ volume standards Val Lead – Jennifer

Howard (and the honorable reviewer of all this data!) The entire DNA/Biology Section for help in laboratory work

and sample donations (you know what I’m talking about!)

Thank you, to Life Technologies/Applied Biosystems for inviting me to speak!

Jarrah R. Kennedy Senior Biology Criminalist

Kansas City Police Crime Laboratory Jarrah.kennedy@kcpd.org

816-349-3237

THANK YOU FOR YOUR ATTENTION!