Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit, presented by Dr. Tomasz Kupiec, Head of the Forensic Genetics Section, Institute of Forensic Research, Krakow, Poland on June 14, 2018.

1. Sample to Insight
Assessment of Y chromosome degradation level using the
Investigator® Quantiplex® Pro RGQ Kit
Dr. Tomasz Kupiec
Head of the Forensic Genetics Section
Institute of Forensic Research, Kraków, Poland

2. Sample to Insight
QIAGEN would like to thank our speaker,
Dr. Tomasz Kupiec, for his presentation.
Disclaimer:
This is a QIAGEN sponsoredwebinar. QIAGEN is not affiliatedwith the Institute of
Forensic Research, Kraków, Poland. The views expressedherein are those of the
speaker, and do not necessarily express the views of QIAGEN.

3. 14 June 2018
Tomasz Kupiec, Andrzej Doniec, Miłosz Januła
Assessment of Y chromosome degradation
level using the Investigator Quantiplex
Pro RGQ kit

4. - Genetic identification of human remains – introduction
- Role of DNA quantification in laboratory workflow
- Degradation and inhibition effects on STR analysis results
- Quantification kit: sensitivity and mixture analysis
- Degradation index predictive value in Y-STR and autosomal STR
DNA analysis
Topics discussed

5. Identification of human remains
• Unidentified bodies
• Disaster victims
• Historical figures
• Archaeological material

6. • Forensic anthropology
• Fingerprints
• Forensic odontology
• Radiology
• Medical data
• Historical data
• DNA testing
Identification methods

7. Identification of historical figures

8. Genetic analysis of putative skull of
Jan Kochanowski (1530-1584)
- detection of SRY sex informative
sequence – Quantifiler Duo,
- amelogenin exon 2/intron 2 sex-
differentiationregion (Genederplex),
- NGM, PowerPlex ESI,
- analysis of Y-STR and X-STR markers,
- mtDNA analysis,
- eyes colour prediction.

9. Victim identification workflow
STAGE A:
Analysis of
archives
STAGE B:
Collecting
reference
STAGE C:
Exhumation work
Examination of
Remains
Examination of
Evidence
STAGE D:
Genetic testing
STAGE E:
Final
identification
Historical data
Anthropological
data
Victim identification workflow
Source: https://www.pbgot.pl

10. Exhumation
Source: https://www.pbgot.pl

11. ExhumationExhumation
Source: https://www.pbgot.pl

12. Exhumation
Source: https://www.pbgot.pl

13. Sample selection
• The hardest part of skeleton
• Positive DNA analysis results:
90-100%
80-90%
70-80%
<70%

14. Laboratory Workflow
Magnetic extraction
Organic extraction
(bones)
Quantification
DNA
Amplification
Electrophoresis
X2
NGS
Source:https://www.thermofisher.com
Source:https://www.qiagen.com
Source:https://www.qiagen.com
Source:https://www.qiagen.com

15. •
Autosomal markers Y Chromosome mtDNA
Genetic markers inheritance

16. 16
NGS results

17. Casework sample analysis – bone samples
17 bone samples with different degradation level
0
20
40
60
80
100
1,00 1,25 1,30 1,50 2,59 3,84 3,97 4,40 5,95 7,99 9,46 10,80 15,61 18,90 22,70 27,00 125,00
%typedmarkers
Degradation Index
samples with DNA input below 0,5ng
Amplification success rate - autosomal markers

18. Casework sample analysis – petrous bone DI=19

19. Casework sample analysis – tooth DI=8

20. Effect of humic acid (HA) - Quantifilier Trio
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
HA 0 HA 50 HA 100 HA 200 HA 400 HA 600 HA 800 HA 1000 HA neg
Humic acid concentration in final volume of qPCR mixture [ng/ul]
DNAquantity[ng/ul]
26
26,5
27
27,5
28
28,5
29
29,5
30
30,5
IPCCtvalue
large
small
IPC
1 2 3
1: 1000ng/ul of HA
2: 1500ng/ul of HA
3: 2500ng/ul of HA
HA concentration
in PCR reaction

21. 1: 1000ng/ul of HA
Inhibition

22. 2: 1500ng/ul of HA
Inhibition

23. 3: 2500ng/ul of HA
Inhibition

24. Investigator Quantiplex PRO RGQ
• Ready to use qPCR real time assay,
• Quantification: total human DNA, male
DNA,
• Information on degradation status:
– Total human DNA,
– Male DNA !!!
• Information about inhibition:
– new Internal Control (length 434 bp = better
correlation with STR results)
• Sensitivity: down to 0.1pg/ul

25. Comparison of the different qPCR
quantification kits
PowerQuant
Investigator
Quantipleks Pro RGQ
Quantifiler Trio
degradati
on
294bp large 353bp large 214bp
human 84bp small 91bp small 80bp
IPC 435bp IPC 434bp IPC 130bp
Y 81bp 136bp Y small 81bp Y 75bp
Y large 359bp

26. Q-Rex software layout – IC analysis

27. Q-Rex software layout – autosomal
DNA analysis

28. Validation experiments
- Sensitivity
- Stability – inhibitors: humic acid (HA)
- Mixture study
- Casework samples analysis – bone samples
- Artificially degraded DNA samples
Source:https://www.qiagen.com

29. Materials and methods – casework
samples
• 80 bone samples with different stage of decomposition
(from 1 to 50 years after death),
– DNA quantity of 13 samples was below 0,001ng/ul
– 13 samples out of 67 were classified as a non-male
material (female )
– 3 samples out of 54 were excluded from experiment
due to quality issues

30. Materials and methods - bones preparation
and extraction
- PrepFiler BTA Forensic DNA Extraction Kit
(Thermo Fisher Scientific)
Source:https://www.thermofisher.com

31. Materials and methods - DNA quantification
- Investigator Quantiplex Pro RGQ Kit
(5 dye detection: green – male small,
yellow – small autosomal, orange – large
male, red – large autosomal,
Crimson- IC)
- Rotor-Gene Q
Source:https://www.qiagen.com

32. Materials and methods – DNA amplification
and electrophoresis
Amplification:
-ESSplex SE QS - 16 STRs + 2 quality markers + Amelogenin (QS1,
Amelogenin, TH01, D3S1358, vWA, D21S11, QS2, D163539, D1S1656,
D19S433, SE33, D10S1248, D22S1045, D12S391, D8S1179, D2S1338,
D2S441, D18S51, FGA)
-YFiler plus – 25 Y-STRs (DYS19, DYS385 a/b, DYS387S1 a/b, DYS389
I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439,
DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518,
DYS533, DYS570, DYS576, DYS627, DYS635 (Y GATA C4), and Y GATA
H4)
Targeted DNA input: 1ng
Source:https://www.thermofisher.com

33. Materials and methods – electrophoresis
Electrophoresis:
- ABI 3500xl
ESSplex SE QS:
- Analytical threshold: 200rfu (based on Qiagen recommendation)
Yfiler Plus:
- Analytical threshold: 334rfu (based on internal validation)
Software:
- GeneMapperID-X ver. 1.5
Source:https://www.thermofisher.com

34. Materials and methods – artificially degraded
DNA and inhibitor resistance
DNA digestion enzyme:
- RNase-Free Dnase I Set
(Qiagen)
DNA input:
- 10ng
- 20ng
Time of digestion:
- 10’
- 20’
Inhibitor:
- Humic acid (Life
Technology)
DNA input:
- 0,1ng
Inhibitor concentration:
- 200 ng/ul
- 400 ng/ul
- 600 ng/ul
- 800 ng/ul
- 1000 ng/ul

35. Comparison of male and female
standard quantification
Name Male/Female Expected
quantity
[ng/ul]
Measured quantity [ng/ul]
Small
(A)
Large
(A)
Small
(Y)
Large
(Y)
IC
[Ct]
R1A* Male 5 Male NIST standard control
R2A Male 0,5 0,4782 0,6466 0,4885 0,4955 15,474
R3A Male 0,05 0,0507 0,0484 0,0467 0,0457 15,978
R4A Male 0,005 0,0013 0,0012 0,0027 0,0025 15,901
R5A Male 0,0005 0,0003 0,0005 0,0002 0,0002 15,832
R1B* Female 5 Female NIST standard control
R2B Female 0,5 0,3915 0,3900 Undetermined Undetermined 15,562
R3B Female 0,05 0,0431 0,0420 Undetermined Undetermined 15,657
R4B Female 0,005 0,0025 0,0028 Undetermined Undetermined 15,663
R5B Female 0,0005 0,0002 Undetermined Undetermined Undetermined 15,712
*NIST DNA standards were not quantified

36. Male/Female and Female/Male
mixtures (2 repeats)
Name Ratio
(M:F)
Expected total
human DNA
quantity [ng/ul]
Measured quantity [ng/ul]
Small
(A)
Large
(A)
Small
(Y)
Large
(Y)
IC
[Ct]
R1A:R1B
1:1 10 9,5196 9,8919 6,1465 6,2870 15,336
R2A:R1B
1:10 5,5 5,0522 4,3373 0,5539 0,5349 15,642
R3A:R1B
1:100 5,05 4,2500 4,1027 0,0694 0,0601 15,832
R1A:R2B
10:1 5,5 5,9531 5,8737 5,6102 5,6977 15,766
R1A:R3B
100:1 5,05 5,8618 5,6726 5,6948 5,3899 15,785
R1A:R1B
1:1 10 10,8358 10,5706 6,3617 6,7402 15,771
R2A:R1B
1:10 5,5 4,7146 4,5717 0,5639 0,5089 15,649
R3A:R1B
1:100 5,05 4,6594 3,8067 0,1001 0,0962 15,620
R1A:R2B
10:1 5,5 5,9160 6,7323 6,2766 5,7118 15,507
R1A:R3B
100:1 5,05 6,1061 7,1755 6,3999 6,1342 15,710

37. Inhibitor resistance: humic acid (HA)
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,18
0,20
16
17
18
19
20
21
Investigator Quantiplex Pro RGQ Kit
large small IPC
HA 200 HA 400 HA 600 HA 800 HA 1000
DNAconcentration
[ng/ul]
ICvalue[Ct]
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,18
0,20
16
17
18
19
20
21
Investigator Quantiplex Pro RGQ Kit
large small IPC
HA 200 HA 400 HA 600 HA 800 HA 1000
DNAconcentration
[ng/ul]
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,18
0,20
16
17
18
19
20
21
Investigator Quantiplex Pro RGQ Kit
large small IPC
HA 200 HA 400 HA 600 HA 800 HA 1000
DNAconcentration
[ng/ul]

38. autosomal [ng/ul] Y chromosome [ng/ul]
repeat
DNA
amount
[ng/ul]
time of
digestion
[min]
small small SD large large SD small
small
SD
large
large
SD
I
1
10 20
0,3003
0,0212
0,0102
0,0006
0,3016
0,0262
0,0069
0,00062 0,2584 0,0092 0,3034 0,0062
0,2527 0,0087 0,2470 0,00543
II
1
20 20
0,8809
0,0350
0,0484
0,0055
0,9008
0,0453
0,0225
0,00142 0,8349 0,0429 0,8943 0,0255
0,7952 0,0350 0,8016 0,02233
III
1
10 10
0,1148
0,0043
0,0036
0,0003
0,1540
0,0082
0,0040
0,00082 0,1112 0,0031 0,1618 0,0038
0,1216 0,0038 0,1739 0,00553
IV
1
20 10
0,3584
0,0068
0,0099
0,0009
0,4277
0,0234
0,0089
0,00042 0,3586 0,0103 0,3711 0,0096
0,3730 0,0120 0,4066 0,00983
DNAse I degraded samples

39. DNAse I degraded samples
0
5
10
15
20
25
30
35
40
45
50
0,0
0,2
0,4
0,6
0,8
1,0
small autosomal DNA concentration [ng/ul] autosomal DNA degradation index Y chromosome degradation index
DNAconcentration
[ng/ul]
Degradationindex
I II III IV

40. Y-STR vs autosomal STR
sample

41. DI values for Y-STRs and autosomal
STRs in bone samples
0
20
40
60
80
100
120
140
160
180
200
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Comparison of degradation indexes
DI of autosomal DNA DI of Y chromosome DNA
DI
DI Y-STRs > DI autosomal STRs
sample

42. Autosomal DNA DI vs STR success
rate in casework samples
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Comparison of degradation indexes and identified loci
(autosomal STRs)
DI of autosomal DNA Investigator ESSplex SE QS
DI
Successrate
DI value >20: loci dropouts
sample

43. 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
160,0
180,0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Comparison of degradation indexes and analysis success rate
(Y-STRs)
DI of Y chromosome DNA Yfiler Plus
DI
Successrate
Male DNA DI values vs Y-STR in
casework samples
DI value > 10: loci dropouts
sample

44. Y-STR raw data (YFiler Plus)
DI: 41,4
Number of loci: 9 out of 25

45. Y-STR (YFiler Plus)

46. Autosomal STR raw data
(ESSplex SE QS )
DI: 27.4
Number of loci : 16 out of 16

47. Autosomal STR (ESSplex SE QS)

48. DI thresholds
Quantifiler Trio
Forensic Science International: Genetics 16 (2015) 77–85
Investigator Quantiplex PRO RGQ
InvestigatorESSplex SE QS
DI > 20 severely degraded
YFiler Plus
DI > 10 severely degraded

49. • Investigator Quantiplex Pro RGQ kit is
particularly useful for the quantification of
challenging samples due to its resistance to
inhibition and high sensitivity,
• Investigator Quantiplex Pro RGQ kit
provides extensive and reliable information
concerning quality of DNA sample (DI for
autosomes and Y chromosome, inhibitor
presence),
• Male DNA degradation index and autosomal
degradation index should be used separately
to predict STR analysis success rate.
Summary

50. The authors would like to thank
Qiagen company for supplying
Investigator Quantiplex Pro
RGQ kit and Investigator
ESSplex SE QS used in this
study.
Special thanks to
Sylwia Chrobak from Qia-Lab
for her support.
Acknowledgment

51. Thank you
tkupiec@ies.krakow.pl

52. Sample to Insight
The Investigator Quantiplex Pro RGQ Kit is intended for molecular biology
applications in forensic, human identity and paternity testing. This product is not
intended for diagnosis, prevention, or treatment of a disease.
Thank you for your attention!
Trademarks: QIAGEN®, Sample to Insight®, Investigator®, Quantiplex® (QIAGEN Group). Registered names,
trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered
unprotected by law.
PROM-12691-001 07/2018© 2018, QIAGEN, all rights reserved.
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