maximizing allele detection by selecting optimal ... · application of thresholds . effect of...

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Maximizing Allele Detection by Selecting

Optimal Analytical Thresholds

Christine A. Rakay

Joli Bregu

Cheng-Tsung Hu

Catherine M. Grgicak

American Academy of

Forensic Sciences February

2013




Steps During DNA Interpretation

Comparison to

Known(s)

Validation

studies &

Literature

Application of

Thresholds

Effect of AT’s on Data Analysis

2 males, 1:19 at 2ng, 1ul 3130 prep volume and 5s injection




Summary of Methods Boston University School of Medicine



ISHI Mixture Interpretation Workshop, 2012. http://www.cstl.nist.gov/strbase/mixture.htm

89% of respondents use an AT between

50 and 150 RFU

Summary of Methods

Method 1.

◦ Kaiser (IUPAC 1976)

Winefordner 1983 and Krane 2007

Method 2.

◦ Currie (IUPAC 1995)

Winefordner 1983

Method 3.

◦ Example in SWGDAM Guidelines

Method 4.

◦ Largest observed noise peak

Method 5.

◦ Miller & Miller. Statistics for Analytical Chemistry (Ellis Horwood & Prentice Hall)

IUPAC 1997 ElectroAnalytical Committee

Method 6.

◦ 1997 IUPAC ElectroAnalytical Committee Recommendations

Use d

ata

fro

m n

egatives (

i.e.

sam

ple

s w

ith n

o D

NA

)

Use d

ata

fro

m

DN

A d

ilution

series




J. Bregu et al. Analytical Thresholds and Sensitivity: Establishing RFU Thresholds for Forensic DNA Analysis. JFS (2013) 1

pg 120-129.

Method 1 to 4 - Negatives Boston University School of Medicine



-Negative sample run with an internal size standard (not shown) using manufacturer’s

recommended protocol

Negative = extraction or amplification negative

15

0

15

0

15

0

15

0

Green and Blue channels seem ‘quieter’ than yellow and red

Baseline is never below 0 RFU Processed data!

Method 5 to 6 – Positives (Standard Curves) Boston University School of Medicine



Regression of positive samples (i.e. single source samples)

Amplified 0.0625-4ng dilution series, injected 5s using manufacturer’s recommended protocol

Plot of Input DNA (ng) versus average peak height (per color) – with error bars

◦ If a peak was homozygous, the RFU was divided by 2

• The points at 2 and 4 ng fall off

the line (PCR efficiency approaching a plateau)

• The error bars become larger with increased DNA input

• A weighted linear regression is within the linear range (i.e. 0.0625 – 1 ng) was used.

Summary of Results

Method Origin

Analytical Threshold for green

5s injection example

1 Negatives 7

2 Negatives 4

3 Negatives 18

4 Negatives 6

5 DNA Series 31

6 DNA Series 39




False non-labeling of alleles (Drop-out) Boston University School of Medicine



Single source 0.125ng, 1ul 3130 prep volume

0

200

40

20

80

Drop-out with Respect to ATs - <0.5 ng DNA Boston University School of Medicine



0

0.2

0.4

0.6

0.8

1

0 50 100 150 200

Fre

qu

en

cy o

f Dro

p-o

ut

Analytical Threhold (RFU)

■ locus DO

■ allelic DO

■ sum (# loci exhibiting

DO)

-As AT increases, locus DO increases, while allele DO stabilizes after 50 RFU then starts to decrease after AT of ~150 RFU. -Although a higher AT (i.e. >150 RFU) begins to decrease the number of loci where allele DO occurs (less stochastic variation),

-Locus DO increases, resulting in an overall increase in DO with AT for Low-template samples

hetlocitotal

alleleDOhetlocilocusfreqDO

#

)2(#)(

hetlocitotal

alleleDOhetlociallelefreqDO

#

)1(#)(




-AT’s have a large effect on the ability to detect/label alleles.

-Red = high level of allele drop-out, blue=low levels of allele drop-out. - To take a ‘conservative’ approach and utilize high AT values leads to a substantial level of Type II errors for low-level samples (i.e. <1000RFU).

Balancing Type I and Type II Errors – < 0.5ng

Impact of ATs on STs Boston University School of Medicine



Analytical

Threshold

(RFU)

Description of

Calculation

Frequency of

Allele Drop-

out

Peak Height of Largest

Surviving Allele at a locus

exhibiting allele drop-out

(RFU)

Frequency of

Locus Drop-

out

Negatives Kaiser’s 0.025 126 0.000

Negatives Kaiser’s 0.041 126 0.002

Negatives Max observed

noise peak 0.168 170 0.028

Positives IUPAC 0.230 240 0.074

50 N/A 0.246 229 0.074

150 N/A 0.246 349 0.593

200 N/A 0.184 548 0.816

N/A = not applicable

500 – 63 pg

Baselines Positives ≠ Baselines Negatives

30

0

30

0

30

0

High

input of

DNA

Neg amp

control

Low

input of

DNA




More on Baselines and Noise Boston University School of Medicine



• This is not instrument baseline/noise

• Single source DNA data amplified from 0.0625 – 2 ng

• Differentiated ‘noise’ from artifact

• -A, pull-up, stutter (+ or -), spikes, dye artifacts

• Plotted RFU of the known/expected peak versus the highest ‘noise’ peak

• High noise with >0.5 ng of DNA, higher AT needed for higher-template samples

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.0 0.1 0.2 0.3 0.4

Pro

po

rtio

n o

f m

ino

r al

lele

lab

led

Proportion of Loci with Noise >AT

Mixture 1:9

> 0.5 ng

<0.5 ng

ATM2

AT= 50

Injection Times Boston University School of Medicine



0

20

40

60

80

100

120

<500 500-1000 1000-1500 1500-2000 2000-2500

Nu

mb

er o

f p

eaks

Avg allele height (RFU)

Amplification positive sample (2 sec)

blue

green

yellow

red

020406080

100120140160180200

Nu

mb

er

of

pe

aks


Amplification positive samples (5 sec)

blue

green

yellow

red

020406080

100120140160180200

<5

00

50

0-1

00

0

10

00

-15

00

15

00

-20

00

20

00

-25

00

25

00

-30

00

30

00

-35

00

35

00

-40

00

40

00

-45

00

45

00

-50

00

50

00

-55

00

55

00

-60

00

60

00

-65

00

65

00

-70

00

70

00

-75

00

Nu

mb

er o

f p

eaks


Amplification positive samples (10 sec)

blue

green

yellow

red

injection times

# of noise peaks

height noise peaks

AT

High-template Low-template

injection times

# of noise peaks

height noise peaks

AT

Conclusions

• Baseline does not remain constant between negatives and samples

with a significant amount of DNA

• There may be amplification ‘noise’ that cannot be characterized as

known artifact (i.e. bleed-through, spike, stutter, etc)

• Optimal ATs will be dependent on the DNA amplification mass

• Optimal AT for DNA samples amplified with < 0.5 ng was 10- 20

RFU.

• An AT of 50 resulted in ~ 20% Type II error rate. An AT of

150 resulted in ~80% error rate.

• To minimize error for DNA samples amplified with > 0.5 ng the AT

needs to be increased by a factor of 2.5 - 5 (i.e. 50 RFU)

• Thresholds designed for/by samples containing optimal masses are not

optimal for low-template DNA interpretation

• Samples amplified with sub-optimal masses require special

interpretation schemes/methods




Acknowledgements

Thanks to the following Boston University BMFS students,

◦ Christine A. Rakay

◦ Joli Bregu

◦ Kevin Hu

◦ Thank-you

◦ Robin Cotton, Charlotte Word, Michael Coble, John Butler, Desmond Lun

◦ Supported by

◦ NIJ2008-DN-BX-K158 training grant awarded by the National Institute of Justice, Office

of Justice Programs, U.S. Department of Justice. The opinions, findings and

conclusions or recommendations expressed in this presentation are those of the

authors and do not necessarily reflect those of the Department of Justice

◦ Boston University, Biomedical Forensic Sciences Program

◦ [email protected]




mailto:[email protected]

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