IDNADEXImproving 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

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 globallyISEC 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

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

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%

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 samplesRepeatability & 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 speciesPCR 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

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

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.

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 RFU AT: 50 RFU

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

3

S

1

3

5

8

v

W

A

D

1

6

S

5

3

9

C

S

F

1

P

O

T

P

O

X

Y

-

I

N

D

E

L

A

M

E

L

D

8

S

1

1

7

9

D

2

1

S

1

1

D

1

8

S

5

1

D

Y

S

3

9

1

D

2

S

4

4

1

D

1

9

S

4

3

3

T

H

0

1

F

G

A

D

2

2

S

1

0

4

5

D

5

S

8

1

8

D

1

3

S

3

1

7

D

7

S

8

2

0

S

E

3

3

D

1

0

S

1

2

4

8

D

1

S

1

6

5

6

D

1

2

S

3

9

1

D

2

S

1

3

3

8

D

3

S

1

3

5

8

v

W

A

D

1

6

S

5

3

9

C

S

F

1

P

O

T

P

O

X

Y

-

I

N

D

E

L

A

M

E

L

D

8

S

1

1

7

9

D

2

1

S

1

1

D

1

8

S

5

1

D

Y

S

3

9

1

D

2

S

4

4

1

D

1

9

S

4

3

3

T

H

0

1

F

G

A

D

2

2

S

1

0

4

5

D

5

S

8

1

8

D

1

3

S

3

1

7

D

7

S

8

2

0

S

E

3

3

D

1

0

S

1

2

4

8

D

1

S

1

6

5

6

D

1

2

S

3

9

1

D

2

S

1

3

3

8

D

3

S

1

3

5

8

v

W

A

D

1

6

S

5

3

9

C

S

F

1

P

O

T

P

O

X

Y

-

I

N

D

E

L

A

M

E

L

D

8

S

1

1

7

9

D

2

1

S

1

1

D

1

8

S

5

1

D

Y

S

3

9

1

D

2

S

4

4

1

D

1

9

S

4

3

3

T

H

0

1

F

G

A

D

2

2

S

1

0

4

5

D

5

S

8

1

8

D

1

3

S

3

1

7

D

7

S

8

2

0

S

E

3

3

D

1

0

S

1

2

4

8

D

1

S

1

6

5

6

D

1

2

S

3

9

1

D

2

S

1

3

3

8

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

170

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 thresholdRFU 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

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

0

0

0

33,33

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

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 %

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)

500:500 - 1500:500 - 2500:500 - 3333:666 - 1333:666 - 2333:666 - 3166:833 - 1166:833 - 2166:833 - 3111:888 - 1111:888 - 2111:888 - 391:909 - 191:909 - 291:909 - 362:938 - 162:938 - 262:938 - 348:952- 1

48:952 - 2 48:952 - 3

1:1

1:2

1:5

1:8

1.10

1:15

1:20

D3S1358

vWA

D16S539

CSF1PO

TPOX

INDEL

AMG

D8S1179

D21S11

D18S51

DYS391

D2S441

D19S433

TH01

FGA

D22S1045

D5S818

D13S317

D7S820

SE33

D10S1248

D1S1656

D12S391

D2S1338

TOTAL DNA INPUT:

1 ng

TOTAL DNA INPUT:

1 ng

1500:1500 - 11500:1500 - 21500:1500 - 3999:1998 - 1999:1998 - 2999:1998 - 3498:2499 - 1498:2499 - 2498:2499 - 3333:2664 - 1333:2664 - 2333:2664 - 3273:2727 - 1273:2727 - 2273:2727 - 3186:2814 - 1186:2814 - 2186:2814 - 3144:2856 - 1144:2856 - 2

144:2856 - 31:20

1:15

1.10

1:8

1:2

1:1

1:5TOTAL 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

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

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

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

Two

Co

ntrib

uto

rs

Th

ree

Co

ntrib

uto

rs

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

Allele Concordance with other Kits (Intra & Inter-lab comparison)

Two

Co

ntrib

uto

rs

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, …)

Allele Concordance with other Kits (Intra & Inter-lab comparisons)

Th

ree

Co

ntrib

uto

rs

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, …)

Non-concordance at SE33 among different Kits (primer designs)

Pro

me

ga

6C

V2

SE33SE33 17 2117 21 28.2 32.228.2 32.2

Glo

ba

lFile

r® k

iit

SE33SE33 17 2117 21 28.2 32.128.2 32.1

Allele 28 at D12S391 (Out of ladder)

Pro

me

ga

6C

V2

D12S391D12S391 D19S433D19S433

Glo

ba

lFile

r® k

it

D12S391D12S391 D2S1338D2S1338

CE Resolution of Microvariants at D12S391

1616

17.3

18

18.3 19.3

Glo

ba

lFile

r® k

it

D12S391D12S391

1616

20

17.3

18

18.3 19.3

Pro

me

ga

6C

V2

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

Minimum Number of Contributors

Minimum Number of Contributors

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 INHIBITORS3 DNA samples with PCR inhibitors showed full Globalfiler Profiles

Case-Type Samples: DNA DEGRADATIONDegraded 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

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

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.

PROJECT TEAM

Pablo Martín

Lourdes Fernández de Simón

Gracia Luque

Mª José Farfán

INTCFINTCFINTCFINTCF----MadridMadridMadridMadrid

SERVICIO DE SERVICIO DE SERVICIO DE SERVICIO 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.

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.