IDNADEX: Improving DNA Data Exchange Validation Studies of a Global STR System Presented by Dr. Antonio Alonso National Institute of Toxicology and Forensic Sciences, Madrid, Spain Concluding Remarks: The data reported demonstrates the GlobalFiler® PCR amplification system followed by CE detection in a 3500 Genetic Analyzer generates high quality, reproducible, precise, accurate, and sensitive profiling STR data, even from sub-nanogram amounts of genomic DNA template and also in the presence of powerful PCR inhibitors co-extracted with DNA from a high variety of forensic samples including the following DNA sources: blood, semen & saliva stains, hair, muscle, liver, bone and teeth. The GlobalFiler® system provides equivalent overall performance to previous forensic STR PCR kits, but with enhanced discrimination power for a better match efficiency that would reduce the chance of adventitious matches during DNA data exchange among national DNA databases. The use of GlobalFiler® kit would be also very beneficial to improve discrimination power of DNA analysis not only in criminal DNA databases, but also in many other forensic applications of autosomal STR profiling including missing person identification, disaster victim identification, mass grave investigations and kinship analysis.

1. IDNADEX
Improving DNA Data Exchange
Validation Studies of a Global STR System
INTCF-Madrid
SERVICIO DE
BIOLOGIA
Supported by a grant from the ISEC programe
Madrid 04/03/2015

2. ESS STR
Core Loci
CODIS
STR Core
Loci
Other
STR Loci
In
Forensic
Kits
Spanish
Database
(200.000
profiles)
STR Markers in
ESS typing kits
CSF1PO CSF1PO
TPOX TPOX
D5S818 D5S818
D7S820 D7S820
D13S317 D13S317
D16S539 D16S539 D16S539
FGA FGA FGA FGA
TH01 TH01 TH01 TH01
VWA VWA VWA VWA
D3S1358 D3S1358 D3S1358 D3S1358
D8S1179 D8S1179 D8S1179 D8S1179
D18S51 D18S51 D18S51 D18S51
D21S11 D21S11 D21S11 D21S11
D22S1045 D22S1045
D1S1656 D1S1656
D10S1248 D10S1248
D2S441 D2S441
D12S391 D12S391
D2S1338 D2S1338 D2S1338
D19S433 D19S433 D19S433
SE33 SE33
Penta D
Penta E
Proposed
21 STR Typing
System
CSF1PO
TPOX
D5S818
D7S820
D13S317
D16S539
FGA
TH01
VWA
D3S1358
D8S1179
D18S51
D21S11
D22S1045
D1S1656
D10S1248
D2S441
D12S391
D2S1338
D19S433
SE33
Proposal: Validation of a single 21 STR typing system
based on 6-Dye CE detection to improve DNA data
Exchange locally and globally
ISEC Programme
Targeted Call 2011
Awarded July 2012
Requirements to the
Companies
21 STR covering ESS &
CODIS + D2, D19 & SE33
Concordance of primer
sequences design with
previous systems
Master mix with high
tolerance to PCR
inhibitor for casework
application

3. Validation Studies on DNA Mixtures Using a 6-Dye System
Containing 23 STR and 3 Y-STR Loci
Pablo Martín, Lourdes Fernández de Simón, Gracia Luque, Mª
José Farfán, Antonio Alonso
Validation Studies on DNA Mixtures Using the PowerPlex®
Fusion 6C System
Promega Webinar Series. Genetic Identity
Original Webinar Date: Thursday, February 5, 2015
Pablo Martín, Lourdes Fernández de Simón, Gracia Luque, Mª
José Farfán, Antonio Alonso
IDNADEX: Improving DNA Data Exchange
FSI Genetics 13 (2014) 68-78

4. Marker Chromosomal
Location
Basic Repeat
motif
Dye
Label
Allele
sizes
(bp)
He DP CE
D3S1358 3p21.31 TCTA/TCTG 6-FAM™ 96-141 0.7993 0.9207 0.6930
vWA 12p13.31 TCTA/TCTG 156-209 0.7817 0.9405 0.4253
D16S539 16q24.1 GATA 227-267 0.7324 0.9061 0.5179
CSF1PO 5q33.3-34 AGAT 282-318 0.7465 0.8754 0.4550
TPOX 2p23-2per AATG 337-377 0.7183 0.8397 0.3189
D8S1179 8q24.13 TCTA/TCTG VIC® 114-171 0.8310 0.9384 0.6578
D21S11 21q11.2-q21 TCTA/TCTG 182-239 0.8451 0.9512 0.6930
D18S51 18q21.33 AGAA 261-342 0.8732 0.9664 0.6930
D2S441 2p14 TCTA NED™ 76-113 0.7641 0.9035 0.5510
D19S433 19q12 AAGG 118-171 0.8345 0.9178 0.6562
TH01 11p15.5 AATG/ATG 179-218 0.7958 0.9179 0.5179
FGA 4q28 CTTT 223-378 0.8697 0.9624 0.7685
D22S104 22q12.3 ATT TAZ™ 88-121 0.6549 0.7782 0.5510
D5S818 5q21-31 AGAT 138-183 0.6901 0.8658 0.3969
D13S317 13q22-31 TATC 149-243 0.7746 0.9253 0.6930
D7S820 7q11.21-22 GATA 262-298 0.7676 0.9397 0.4859
SE33 6q14 Complex AAAG 307-428 0.9577 0.9790 0.8458
D10S1248 10q26.3 GGAA SID™ 85-129 0.7500 0.9078 0.5179
D1S1656 1q42.2 TAGA 160-207 0.8944 0.9626 0.7685
D12S391 12p13.2 AGAT/AGAC 216-268 0.8838 0.9619 0.7305
D2S1338 2q35 TGCC/TTCC 281-349 0.8732 0.9629 0.6202
General characteristics of autosomal STR markers included in the Globalfiler Kit
and forensic biostatistical parameters in the Spanish population (N=284)
Garcia et al. Forensic Sci Int Genet. 6(2) (2012)
Additional Sex markers of Globalfiler System
Y indel Yq11.221 VIC 81-86 bp
Amelogenin X: p22.1-22.3 ; Y: p11.2 98-104 bp
DYS391 Yq11.21 364-389 bp
Multiplex STR System Probability of Identity Probability of Paternity
Exclusion
NGMSElect 2.5 X 10 -21 99.999998%
Globalfiler 3.1 X 10 -26 99.99999995%

5. Validation Parameters Type & Number of Samples Analyzed
Analytical Threshold 22 PCR negative controls
Sensitivity & Stochastic Threshold 3 Human genomic DNA samples of known human DNA concentration
(SRM-2372A, DNA Control 007 & 1 genomic DNA sample from an
anonymous male donor)
(84 samples: Quadruplicate analysis of 7 dilution series)
Precision and Accuracy 24 injections of the Globalfiler allelic ladder
90 human genomic DNA samples
Repeatability & reproducibility 3 Human genomic DNA samples of known STR profile
(30 samples: Quintuplicate analysis of 5 replicates by two operators)
Heterozygous balance 85 Human genomic DNA samples
Stutter Threshold 85 Human genomic DNA samples
Genotype concordance 80 Human genomic DNA samples from saliva swabs of anonymous donors
10 genomic DNA samples of the DNA profiling standard SRM-2391b
DNA mixtures 1 male /female DNA mixture in 7 different ratios (1 ng of total DNA)
1 male /female DNA mixture in 7 different ratios (3 ng of total DNA)
(42 samples: Triplicate analysis of 7 dilution series)
15 DNA mixture samples from proficiency exercises
Species specificity 21 genomic DNA from human–associated microbial species
PCR reduced reaction volume 10 human genomic DNA samples of known STR profile
Stability study in Case-Type
Samples
54 forensic DNA samples previously analyzed with different STR Kits
Validation Parameters & Materials

6. Methods
Forensic samplesReference samples
DNA ExtractionDNA Extraction
Real time PCR Quantifiler® Duo Kit
Targets: RPPH1 , SRY & IPC
DNA QuantificationDNA Quantification
1 X 95ºC for 60 s
29 X 94ºC for 10 s 59ºC for 90 s
1X 60ºC for 10 min
PCR AmplificationPCR Amplification
Sample Injection: 15 s at 1.2kv
CE: 1550 s at 13 kv
CE Detection 3500 Genetic AnalyzerCE Detection 3500 Genetic Analyzer
GlobalFiler® User guide
Peak Amplitude 50 & 100 RFU
Data AnalysisData Analysis
Management of samples, analysis
and results
Comparison & Concordance studies

7. GlobalFiler® kit profile from a reference saliva swab
(DNA input: 500 pg; PCR Reaction volume :12.5 ul)
Performance of PCR (peak height) under reduced reaction
volume (12,5 µl) in comparison with the standard PCR
reaction volume (25 µl) from a set of 10 human genomic
DNA from saliva reference samples.

8. Blue Green Yellow Red Purple
Average RFU 5,35 10,94 5,71 8,68 9,72
Standard deviation 2,67 3,61 2,30 3,01 3,19
Max. RFU 50 48 53 45 47
Min. RFU 1 2 1 2 2
Average RFU + 3
Standard deviation
13,35 21,77 12,61 17,71 19,28
Average RFU + 10
Standard deviation
32,04 47,02 28,73 38,79 41,58
2 X (Ymax - Ymin) 98 92 104 86 90
Range of analysis: 70-450 bpRange of analysis: 70-450 bp
Analytical Threshold : 22 PCR negative controls
AT: 100 RFUAT: 100 RFU
AT: 50 RFUAT: 50 RFU

9. Sensitivity study & stochastic threshold: Heat maps summarizing the sensitivity
results of the last five DNA serial dilutions (from 250 to 15.25 pg)
250-1
250-2
250-3
250-4
125-1
125-2
125-3 230
125-4
62.5-1
62.5-2 161 138 530
62.5-3
62.5-4 281 186 205
31.25-1 149 103 278 375 130 127
31.25-2 183 353 142 112 194 599 214 157
31.25-3 111 233 335 146 126 201 305 765
31.25-4 168 223 203 212 916 193
15.5-1 168 117 179 203 129 187 114 230 237 133 135 120
15.5-2 171 136 187 266 290 107 162 210 148
15.5-3 114 168 228 132 126 113
15.5-4 286 173 279 123 119 113 103 160 274
811
313
542
228
110 202 120 342 191 225 190 264
166 143 102 194 321 102
117 139 239 105 127 267
122 149 400 170 127 205
278 139 508 281 190 163 314 180 257
135 104 118 194 226 145 172 249 136 169 214
114 114 222 148 237 156 115 120 175 181
154 143 254 135 142 196
162
661 158
102
165 121
121 176 172
342 476 354 166
101 170 622
198 174 154 393 144 110 207 147 113
113 150 440 152 394
226 424 131
131 212 184 158 553 158
2372 A Control 007 Genomic DNA
D
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100RFU100RFU
250-1
250-2
250-3
250-4
125-1
125-2
125-3 230
125-4
62.5-1
62.5-2 161 530
62.5-3
62.5-4 205
31.25-1 149 56 130 127
31.25-2 183 353 599 157
31.25-3 51 233 126 765 66
31.25-4 223 81 86 212 916 193
15.5-1 179 203 129 81 187 94 53 114 230 135 120
15.5-2 114 266 162 210 86 62
15.5-3 114 55 168 132 126 113 55 95
15.5-4 173 279 123 119 103 92 160 274
110 202 54 342 191 190 72 90
166 65 60 194 55 92 84
89 77 117 91 105 72 127 74
122 75 85 400 86 170 76 127 62 205
278 60 85 63 508 190 82 163 314 180 257
118 226 145 54 172 249 136
87 114 53 222 148 53 115 120 86 83 52 175 181
55 154 59 50 143 57 71 196 59 51
102
121
121
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154 83 113
113 150 152 394
60 226 88
97 158 553 158 58
2372 A Control 007 Genomic DNA
50 RFU50 RFU
Stochastic threshold
RFU value of the highest surviving false homozygous peak when allele drop-out is observed at heterozygous markers:
916 RFU for D12S391 at 31.25 pg of DNA input

10. 12,84
2,77
0
0
0
30,9
19,09
1,04
0,34
0
56,25
78,12
98,95
99,65
100
0 20 40 60 80 100
15,5
31,25
62,5
125
250
29,51
14,24
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20,83
4,86
0,34
0
37,15
67,01
94,44
99,65
100
0 20 40 60 80 100
15,5
31,25
62,5
125
250
100RFU100RFU 50 RFU50 RFU
Overall percentage results (SRM-2372A, DNA Control 007, and the genomic DNA
from an anonymous donor) of full allele profiles , allele drop-out , and locus drop-out

11. Sensitivity study: Incidence of Drop-in at Low DNA input
D2S441-007-15 PGD2S441-007-15 PG
69
RFU
69
RFU
D7S820-007-62 PGD7S820-007-62 PG
113
RFU
113
RFU
D22S104-007-62 PGD22S104-007-62 PG
66
RFU
66
RFU
TPOX-007-62 PGTPOX-007-62 PG
84
RFU
84
RFU
D21S11-007-125 PGD21S11-007-125 PG
84
RFU
84
RFU
D3S1358-2372A-125 PGD3S1358-2372A-125 PG
63
RFU
63
RFU
D8S1179-2372A-125 PGD8S1179-2372A-125 PG
100
RFU
100
RFU
D2S441-2372A-250 PGD2S441-2372A-250 PG
59
RFU
59
RFU
SE33-2372A-250 PGSE33-2372A-250 PG
191
RFU
191
RFU
D8S1179-DONOR-31 PGD8S1179-DONOR-31 PG
103
RFU
103
RFU
TH01-DONOR-62 PGTH01-DONOR-62 PG
80
RFU
80
RFU
SE33-DONOR-250 PGSE33-DONOR-250 PG
155
RFU
155
RFU
Detected Incidence of Drop-in over a
threshold of 100 RFU
(1,764 Determinations)
0.28 %
Detected Incidence of Drop-in over a
threshold of 50 RFU
(1,764 Determinations)
0.68 %

12. Species specificity against genomic DNA samples from several human–associated microbial species.
(Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae (serogroup B), Staphylococcus aureus, Escherichia coli, Bordetella pertussis, Bordetella
parapertussis, Alcaligenes faecalis, Acinetobacter calcoaceticus, Flavobacterium odoratum, Morganella morganii, Pseudomonas stutzeri, Providencia stuartii, Proteus
mirabilis, Citrobacter freundii, Pseudomonas aeruginosa, Salmonella enteritidis, Neisseria lactamica, Shigella flexneri, and Serratia marcencens)

13. 500:500 - 1
500:500 - 2
500:500 - 3
333:666 - 1
333:666 - 2
333:666 - 3
166:833 - 1
166:833 - 2
166:833 - 3
111:888 - 1
111:888 - 2
111:888 - 3
91:909 - 1
91:909 - 2
91:909 - 3
62:938 - 1
62:938 - 2
62:938 - 3
48:952- 1
48:952 - 2
48:952 - 3
1:1
1:2
1:5
1:8
1.10
1:15
1:20
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TOTAL DNA INPUT:
1 ng
TOTAL DNA INPUT:
1 ng
1500:1500 - 1
1500:1500 - 2
1500:1500 - 3
999:1998 - 1
999:1998 - 2
999:1998 - 3
498:2499 - 1
498:2499 - 2
498:2499 - 3
333:2664 - 1
333:2664 - 2
333:2664 - 3
273:2727 - 1
273:2727 - 2
273:2727 - 3
186:2814 - 1
186:2814 - 2
186:2814 - 3
144:2856 - 1
144:2856 - 2
144:2856 - 3
1:20
1:15
1.10
1:8
1:2
1:1
1:5
TOTAL DNA INPUT:
3 ng
TOTAL DNA INPUT:
3 ng
DNA mixtures series: Relative ratios of 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, and 20:1 of female / male

14. Full profile of the minor male contributor: Series of 1 ng of total DNA at 5:1 mixture ratio
DNA input of the minor male contributor: 166 pgDNA input of the minor male contributor: 166 pg

15. Full profile of the minor male contributor: Series of 3 ng of total DNA at 10:1 mixture ratio
DNA input of the minor male contributor: 273 pgDNA input of the minor male contributor: 273 pg

16. Body fluid mixture samples from GHEP & GEDNAP proficiency exercises
DNA SAMPLES BODY FLUIDS &
CONTRIBUTORS
[DNA]
ng/ul
DNAMIX01-L1 Total: 5.7
Male: 0.8
DNAMIX01-L2 Total: 0.7
Male: 0.4
DNAMIX01-L3 Total: 0.7
Male: 0.6
DNAMIX02-L1 Total: 13
Male: 15
DNAMIX03 Total: 4
Male: 2
DNAMIX04 Total: 13
Male: 8
DNAMIX05 Total: 9
Male: 3
DNAMIX06 Total: 1.7
Male: 1
DNAMIX07 Total: 3.3
Male: 1.8
DNAMIX08 Total: 1.9
Male: 1.8
DNAMIX09 Total: 1.7
Male: 1.3
DNAMIX10 Total: 3
Male: 0
SEMENSALIVA BLOOD
TwoContributors
ThreeContributors
DNA
SAMPLES
BODY FLUIDS &
CONTRIBUTORS
[DNA]
ng/ul
DNAMIX11 Total: 7
Male: 3
DNAMIX12 Total: 1.6
Male: 1.7
DNAMIX13 Total: 3.5
Male: 1.7
DNAMIX14 Total: 1.7
Male: 0.8
http://www.gednap.de
http://www.gep-isfg.org
Allele Concordance with other kits
MNC

17. Allele Concordance with other Kits (Intra & Inter-lab comparison)
TwoContributors
DNA
SAMPLES
BODY FLUIDS &
CONTRIBUTORS
Allele Concordance
(Globalfiler, Identifiler , NGMSE, ESX17,…)
DNAMIX01-L1 OK
Partial DNA profile from the minor male
contributor at a 7:1 DNA ratio
DNAMIX01-L2 OK
DNAMIX01-L3 OK
DNAMIX02 OK
DNAMIX03 OK
Non-concordance at SE33 among different Kits
(primer designs)
DNAMIX04 OK
DNAMIX05 OK
Allele 28 at D12S391 (Out of ladder)
DNAMIX06 OK
DNAMIX07 OK
DNAMIX08 OK
DNAMIX09 OK
DNAMIX10 OK
(Identifiler, NGMSe, PPESX17, Fusion 6C, …)

18. Allele Concordance with other Kits (Intra & Inter-lab comparisons)
ThreeContributors
DNA
SAMPLES
BODY FLUIDS &
CONTRIBUTORS
Allele Concordance
(Globalfiler, Identifiler , NGMSE, ESX17,…)
DNAMIX11 OK
CE Resolution of Microvariants at D12S391
DNAMIX12 OK
CE Resolution of Microvariants at D12S391
DNAMIX13 OK
DNAMIX14 OK
(Identifiler, NGMSe, PPESX17, Fusion 6C, …)

19. Non-concordance at SE33 among different Kits (primer designs)
Promega6CV2
SE33SE33 17 2117 21 28.2 32.228.2 32.2
GlobalFiler®kiit SE33SE33 17 2117 21 28.2 32.128.2 32.1

20. Allele 28 at D12S391 (Out of ladder)
Promega6CV2
D12S391D12S391 D19S433D19S433
GlobalFiler®kit D12S391D12S391 D2S1338D2S1338

21. CE Resolution of Microvariants at D12S391
1616
17.3
18
18.3 19.3
GlobalFiler®kit
D12S391D12S391
1616
20
17.3
18
18.3 19.3
Promega6CV2
D12S391D12S391
20.3
21
1515 16 20
D12S391D12S391
1515 16 20
21
D12S391D12S391
20.3
Automatic allele calling of 20.3 By GMIDX reanalysis with
No Smoothing
Automatic allele calling of 20.3 By GMIDX reanalysis with
No Smoothing

22. Minimum Number of Contributors

23. Minimum Number of Contributors

24. Full GlobalFiler® profile example from a DNA extract with strong PCR
inhibitors
Sample: Epithelial cells in a glove
QFDUO Total: 0
QFDUO Male: 0
IPC: Undetected
Identifiler® kitIdentifiler® kitIdentifiler® kit GlobalFiler® kitGlobalFiler® kitGlobalFiler® kit
Case-Type Samples: PCR INHIBITORS
3 DNA samples with PCR inhibitors showed full Globalfiler Profiles

25. Case-Type Samples: DNA DEGRADATION
Degraded DNA obtained from 8 bone samples and 5 teeth samples (with a variable post-mortem interval from 3 month to 41 years)
Sample: Pars Petrosa
QFDUO Total: 0.0 25
QFDUO Male: 0
IPC: 29.7
Partial GlobalFiler® profile example from a 41 years old bone sample
MODERATE DNA DEGRADATION

26. NGM SElect™ kitNGM SElect™ kit GlobalFiler® kitGlobalFiler® kit
Sample: FFPE
QFDUO Total: 0,0 4
QFDUO Male: 0.03
IPC: 30
Partial NGM SElect™ & GlobalFiler® profile examples from a
severely degraded DNA sample
Case- Type DNA Samples: HIGH DNA DEGRADATION
FORMALIN FIXED BIOPSIES

27. Concluding Remarks
The data reported demonstrates the GlobalFiler® PCR amplification system followed by
CE detection in a 3500 Genetic Analyzer generates high quality, reproducible, precise,
accurate, and sensitive profiling STR data, even from sub-nanogram amounts of genomic
DNA template and also in the presence of powerful PCR inhibitors co-extracted with
DNA from a high variety of forensic samples including the following DNA sources: blood,
semen & saliva stains, hair, muscle, liver, bone and teeth.
The GlobalFiler® system provides equivalent overall performance to previous forensic STR
PCR kits, but with enhanced discrimination power for a better match efficiency that
would reduce the chance of adventitious matches during DNA data exchange among
national DNA databases.
The use of GlobalFiler® kit would be also very beneficial to improve discrimination power
of DNA analysis not only in criminal DNA databases, but also in many other forensic
applications of autosomal STR profiling including missing person identification, disaster
victim identification, mass grave investigations and kinship analysis.

28. PROJECT TEAM
Pablo Martín
Lourdes Fernández de Simón
Gracia Luque
Mª José Farfán
INTCFINTCFINTCFINTCF----MadridMadridMadridMadrid
SERVICIO DESERVICIO DESERVICIO DESERVICIO DE
BIOLOGIABIOLOGIABIOLOGIABIOLOGIA
BIOLOGY DEPARTMENT STAFF
Acknowledgements
Special thanks also to the staff of our microbiology laboratory for providing the microbial genomic
DNA samples used in the specificity study.
Oscar Garcia
Biostatistical Calculation with the Spanish Population Data
This project has been funded with support from the European Commission (grant
HOME/2011/ISEC/AG/PRUM/4000002125 under the Prevention of and Fight
against Crime (ISEC) programme of the European Commission-Directorate General
Justice and Home Affairs). This publication reflects the views only of the authors,
and the European Commission cannot be held responsible for any use which may
be made of the information contained therein.

29. Speaker was provided travel and hotel support by Thermo
Fisher Scientific for this presentation, but no remuneration.
GlobalFiler® Express PCR Amplification system is For Forensic
and Paternity Use Only. Applied Biosystems™ 3500 Series
Genetic Analyzers are For Research, Forensic or Paternity Use
Only. Not for use in diagnostic procedures.
Life Technologies and its affiliates are not endorsing,
recommending, or promoting any use or application of Life
Technologies products presented by third parties during this
seminar. Information and materials presented or provided by third
parties are provided as-is and without warranty of any kind,
including regarding intellectual property rights and reported
results. Parties presenting images, text and material represent they
have the rights to do so.