quantifiler® trio kit and forensic samples management: a matter of degradation

Human Identification Solutions Conference, Mar 4 2015, Madrid

Quantifiler® Trio kit and forensic samples management:

a matter of degradation

Stefano Vernarecci

Forensic Genetics Laboratory

Scientific Police Service

Department of Ministry of Interior

1

2

Turin

Naples

Rome

Palermo

Forensic Genetic Laboratory of Rome

Scientific Police Service – Central Direction for the Anticrime Police

The Forensic Genetic Laboratories of the Italian National Police

3

“Obtaining a partial DNA profile is analogous to only havinga portion of a phone number. A full phone number might be 001-301-975- 4049. If you only had ‘ 4049, ’ this information would be of limited value since it could match to other phone numbers fromdifferent area codes. However, in some situations, a few digits may be enough information to narrow down the possibilities in aninvestigation. Likewise, while full DNA profiles are preferable, partial profiles may be helpful in some instances.”

J.M. Butler

From “Advanced topic in Forensic DNA Typing: Methodology” J.M. Butler

Degraded samples: an issue for forensic genetic analysis

«ski slope pattern»

4

Sample

Collection

DNA

EXtractionQuantitation

Amplification

of STR

markers

C.E. Data Analysis

� Time consuming

� Money consuming

� Sample consuming

Degraded samples: an issue for forensic genetic analysis

Mini STR (< 200bp)

Conbine DifferentSTR- Kits

Increase Input DNA amount

5

Quantifiler® Trio DNA Quantification Kit

Degradation Index =Small autosomal target DNA conc (ng/µL)

Large autosomal target DNA conc (ng/µL)

The DI represents a general indicator of the level of degradation in the

sample where the higher the DI the greater the degradation.

A DI = 1 should indicate absence of degradation.

… provide information about both concentration and quality of a sample in terms of the

presence of inhibitors and degradation

6

Quantifiler® Trio DNA Quantification Kit

� Evaluate the reliability of the Quantifiler® Trio kit in providing information

on the level of degradation in a sample.

� Study the opportunity to use this information in predicting the quality of

the profile obtained after genotyping.

� Define operative implications, taking into account the DI, to apply to our

routine analysis in order to choose the best PCR strategy when dealing with

degraded samples

7

Quantifiler Duo (probe -140bp) Quantifiler Trio (small Probe Data – 80bp)

Totally 181 forensic samples from adjudicated casework (oral swab, blood, touch DNA, bones, sperm,

hairs ), previously analyzed with Quantifiler Duo were selected on the basis of the obtained profile,

including single source samples and mixture with distinguishable major contribuitor, showing different

levels of degradation.

133

62

52

510- Undeterminated

< 1 Pg/uL

1 Pg - 20 Pg/uL

20-100 Pg/uL

> 100 Pg/uL

2

10

51

42

76

0 - undetermined

< 1 Pg/uL

1 Pg - 20 Pg/uL

20-100 Pg/uL

> 100 Pg/uL

Comparison between Quantifiler® Trio and Quantifiler Duo quantitation data

8

0,00001

0,00010

0,00100

0,01000

0,10000

1,00000

10,00000

100,00000

0 10 20 30 40 50

Samples at increasing concentration

Small and Large autosomal amplicons ( Degradation Index < 1,5)

0,00001

0,00010

0,00100

0,01000

0,10000

1,00000

10,00000

0 10 20 30 40 50 60

Samples at increasing concentration

Small and Large autosomal amplicons ( Degradation Index > 4)

Log

(1

0)

of

Co

nce

ntr

ati

on

s (N

g/m

l)

Quantifiler® Trio Large and Small probe values and the Degradation Index

Overall we observed DI values ranging from a minimum of 0.47 to a maximum of 158

9

Analysis of degradation pattern and comparison with DI parameter

…sometimes appearances could be

deceptive…..

Is the DI parameter obtained during the quantification reliable in providing information

about the degradation in a sample?

Could it be used to predict the quality of a profile in term of degradation after PCR?

10

STR analysis with the GlobalFiler® PCR Amplification kit

0.5 ng of input

DNA (SA probe)

A subset of 95 samples were amplified with the GlobalFiler® PCR Amplification kit using 0.5 ng of input DNA as

established after our internal validation.

11


78-95 bp 204-224 bp

Mean PeakHeight(80)

Mean PeakHeight(214)= 3.8

y = 1,4602x + 1,2875

R² = 0,8201

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18

DI

pH

(80

)/p

H(2

14

)

Samples Mean PH (80)

+/- SD

Total of sample 1944 +/- 664

Not-degraded

samples2041 +/- 704

Degraded

samples1799 +/- 553

peak heights from

homozygous loci

were divided by 2

12

Log H(bp) = log (c) + log (p) bp = α0 + α1 bp

P =exp(α1)

The level of degradation is represented by the P value . It is calculated as the exponential function of the

angular coefficient of the regression line obtained by plotting the logarithm of observed peak height

against the fragment length. P is ≈ 1 for not-degraded sample, decreasing at an increase of degradation.

In the author’s experience P ≈ 0.98 represents a moderately degraded sample.


Drop-out probability and degradation level in a sample

can be estimated based on a log-linear relationship

between Bp and H(bp).

13

y = -0,0103x + 8,4308

R² = 0,8645

4

4,5

5

5,5

6

6,5

7

7,5

8

70 120 170 220 270 320 370 420

CSF1PO,D16S539;D3S1358,TPOX,vWA,AMEL,D18S1179,D10S1248,D12S391,D1S1656,D2S1338,D13S317,

D22S1045,D5S818,D7S820,SE33

D19S433, FGA, TH01

Log

pe

ak

he

igh

t

Size (bp)

• peak heights from homozygous

loci were divided by 2

• The loci DYS391, Y-Indel due to

their male specificity.

• The Loci TH01, FGA and

D19S433 were excluded from

the analysis because showing

generally higher peak heights

than the rest of the profile

Sample’s data fitting to the model was verified by graphical diagnostic and R2 statistic for the log-linearity

as suggested by the model. Samples with R2 value less than 0.7 were excluded from comparison with DI

parameter. Notably most of analyzed samples achieved R2 value more than 0.8.

P= exp (-0.0103)= 0.99


DI= 3.335

GlobalFiler®, 0.5 ng DNA

14

y = -0,0008x + 0,9909

R² = 0,7006

0,97

0,975

0,98

0,985

0,99

0,995

0 5 10 15 20 25

P

DI

DI comparison with P value calculated by logit-regression model

Together these results suggest that the DI parameter, obtained after the Quantification,

could be reliably used to predict the quality of the profile in term of degradation

15

Degradation Index Degradation

< 1.5 No degradation

1.5 – 4 Mild Degradation

4 – 10 Degradation

> 10 Severe degradation

Four arbitrary categories of degradation were proposed …

16

Analysis of the locus drop-out for different degradation categories P

erc

en

tag

e o

f m

ark

ers

ca

lle

d a

bo

ve A

T

No degradation

(DI <1.5)

Mild degradation

(DI >1.5 and <4)

Degradation

(DI >4 and <10)

Severe degradation

(DI >10)

Markers in the plot

are ordered at

increasing of average

alleles length

non-normalized

samples (Input

DNA <0.5 Ng and

> 45 Pg

* * *

17

Pe

rce

nta

ge

of

ma

rke

rs c

all

ed

GlobalFiler® Kit

(Input DNA 1 ng)

GlobalFiler® Kit

(Input DNA 0,5 ng)

No degradation

(DI <1.5)

Mild degradation

(DI >1.5 and <4)

Degradation

(DI >4 and <10)

Severe degradation

(DI >10)

Comparing the performance of different PCR protocols

NGM-SElect™ Kit

(Input DNA 0.5 ng)

18

0

5

10

15

20

25

0,4 0,8 1,6 3,2 6,4 12,8 25,6 51,2

GlobalFiler® (Input DNA 0,5 ng)

GlobalFiler® (Input DNA 1 ng)

NGM-Select ™(Input DNA 0,5 ng)

Log (10) DI

Number of Loci

called

Comparing the performance of different protocols

19

2,20247E-28

5,20187E-23

7,95E-16

1,67E-10

1E-28

1E-26

1E-24

1E-22

1E-20

1E-18

1E-16

1E-14

1E-12

1E-10

1E-08

0,000001

0,0001

0,01

1

GF 1ng

GF0.5 ng

NSE 0.5 ng

DI 0-1.49 DI 1.5-3.99 DI 4-9.99 DI > 10

Average Random Match Probabilities (RMPs)

Analysis of the power of discrimination

The Random Match Probability was calculated as recommended from ISFG, for each profile obtained

from the samples that was possible to amplify with all the three protocols

20

In case of degraded samples concentrated less than 3 Pg/µL No Profile or a maximum of 3

markers are expected to be typed

Concentration

< 33,3 pg/µµµµl and >3

Pg/µµµµL

Concentration

> 33,3 pg/µµµµl

No Degradation.

DI: < 4

FULL or PARTIAL PROFILE

Expected

GlobalFiler® or other STR-Kit

with large volume of Input DNA

FULL PROFILE Expected

GlobalFiler® (0.5 ng), NGM-SE or other

STR kits

Degradation.

DI: > 4

PARTIAL or NO profile Expected

Mini STR or other STR kits for

exclusion purposes

PARTIAL PROFILE Expected

GlobalFiler® (1 Ng of Input DNA if

possible) or other STR kits

… some possible operative implications in managing forensic samples taking into account

the Degradation Index in order to maximize allele recovery

21

DNA

EXtractionQuantitation

Sample

Collection

Increase Input DNA

amount

Mini STR (< 200bp)

Conbine Different

STR- Kits

Amplification

of STR

markers

C.E.Data

AnalysisProfile

To Conclude….

SNPs and NGS

technology

22

Thank You !

Alessandro Agostino

Laura Sard

Lisa Calandro

Daniela Stradiotto Director of Scientific Police Service

Egidio Lumaca Director of III Division

Paola Montagna Forensic Genetics Laboratory Chief

Elisabetta Mei Responsible of Validation

Enrica Ottaviani

[email protected]

Speaker was provided travel and hotel support by Thermo Fisher Scientific for

this presentation, but no remuneration.

Quantifiler® Trio DNA Quantification Kit is For Research, Forensic and Paternity Use

Only. Globalfiler® PCR Amplification kit and NGM SElect PCR Amplification kit are For

Forensic or Paternity Use Only.

quantifiler® trio kit and forensic samples management: a matter of degradation

Technology