1

The Armed Forces DNA Identification LaboratoryJennifer O’Callaghan

University of North Texas –Center for Human Identification

Forensic Science Training and Development Workshop

8 July 2008

Jennifer O CallaghanMitochondrial DNA Analyst II

Armed Forces DNA Identification Laboratory

Gillette Complex(AFDIL)

UPS Complex(AFRSSIR)

DNA Reference Collection And Storage For Department of Defense Personnel

And Other Eligible Individuals

Worldwide Scientific Consultation, Research, And Education Services

In The Field Of Forensic DNA Analysis For The Department of

Defense

Mission

2

AccreditationAmerican Society of Crime Laboratory Directors(ASCLD - LAB Certification)

College of American PathologistsPathologists(CAP Basic Laboratory and Molecular Biology)

DoD DNA Oversight Committee(Part of DoD QA Program (DSB 1995) and reports to ASD(HA)

AFIP Scientific Advisory BoardFuture: National Association of Medical Examiners (NAME)

American Association of Blood Banks (AABB)

AFRSSIRAFRSSIRnucnucDNADNA mtmtDNADNA ResearchResearch

QA/QC & SafetyQA/QC & Safety

BioinformaticsBioinformatics2 153

2 6 1

4

11 3

2

7

1

DoD DNA RegistryDoD DNA Registry

Team 1Team 1ITIT ITITITIT

136 Total Staff

ITITGrantsGrantsTeam 1Team 1

Auto/BiometricsAuto/Biometrics

3

11

Validation/QCValidation/QC8

5

Resource Management

Resource Management

Team 2Team 2

Team 3Team 3Team 4Team 4

Team 5Team 5

Team 6Team 6

Team 1Team 1

Team 2Team 2

Team 3Team 3

43Team 1Team 1

Team 2Team 29

Staffing List:1 Army 052 GS-141 GS-121 GS-111 GS-90 GS-81 GS-72 Contract (EDS)12 Contract (FTI)113 Contract (ARP)

• All Branches of the Armed Forces & Coast Guard• Includes Reserve, National Guard, & DoD Civilians• Collections Started June 1992• As of 31 May 2008: 5,352,393• Total for FY 2007: 271,760

Department of Defense DNA COLLECTION PROGRAM

ota o 00 , 60• Collections at 1,172 Sites• Has Been Used In 4,000 Death

Investigations Since 1992

3

Deoxyribonucleic Acid (DNA)

Information Is Encoded In TheFour Chemical Building Blocks

Of DNA (Bases)

A

T

G

C

T

A

T

A

A

T

A

T

C

G

C

G

T

A

G

C

G

C

C

G

C

G

C

G

T

A

T

A

A

T

G

C

A

T

A

T

T

A

A (adenine) T (thymine)G (guanine) C (cytosine)

23 Pairs of Chromosomes• Chromosomes Contain approximately 30,000 Genes• Made Up of 3.2 Billion Base Pairs of DNA1 22 33 4 5 6

9 10 11 12

13 14 15 16 17

19 20 21 22

77 8

18

23XX

or

XY Chromosome 23

FemaleMale

10,000X magnification of X and Y chromosomes

Types of DNA

Nuclear DNA (nucDNA)

Mitochondrial DNA (mtDNA)

Y-chromosomal DNA (yDNAyDNA)

Slide by S.M. Edson

4

mtDNAMale and Female

Maternal inheritance

Genetic Transmission•Each type is transmitted through the generations by different mechanisms

•Allow for different types of references (Direct vs. Indirect)

•mtDNA and yDNA are NOT Unique to an individual, but to a family line

nucDNAMale and Female

Both parents

yDNAyDNAMale only

Paternal inheritance

AFDIL Support Of The Armed Forces Medical Examiner

• Recent Death Investigations• Potentially Fragmented• Direct References Available

nucDNA Section

DNA As Only Source OfIdentification STAT Case

• No teeth present, no fingerprint available

• Highly fragmented remains

• DNA Reference card for the individual available

• Comparison of DNA profile from reference to sample(s)

• Identification performed expeditiously < 48 hrs.

5

Re-Association Performed As A STAT Case

• No teeth present, no Ante or Post Mortem Fingerprint available

• Highly fragmented remains

• DNA Reference card for the individual available

• Comparison of DNA profile from reference to sample(s)

• Identification performed expeditiously < 48 hrs.

Total Identifications20 March 2003 – 31 April 2008

4,875 - Casualties Processed at the Dover AFB Port Mortuary

• 3,894 Identifications by DNA and either Fingerprint or Dental• 235 Identifications by Fingerprints Only• 53 Identifications by Dental Only• 232 Identifications by DNA Only

Over 15,434 samples total

1396 samples in FY20032414 samples in FY20043069 samples in FY2005 3041 samples in FY20064715 samples in FY2007799 samples in FY2008

99% success rate

USS Cole: Oct 2000USS Cole: Oct 2000

EA 990: Oct 1999EA 990: Oct 1999

Evidence Collection and DNA Sampling

Evidence Collection and DNA Sampling

AKA 261: Jan 2000AKA 261: Jan 2000

UA 93: Sep 2001UA 93: Sep 2001

AA 77: Sep 2001AA 77: Sep 2001Learjet: Oct 1999Learjet: Oct 1999

US Air Express 5481: Jan 2003 US Air Express 5481: Jan 2003

6

Space Shuttle Columbia

Feb 1, 2004

Hurricane Katrina - 29 August 2005Support For The FEMA Disaster Mortuary Operations

Response Teams (DMORT), St. Gabriel, LA & Gulfport, MS

DMORT-West, St. Gabriel, LAAs of 20 March 2006

• 854 Recovered Remains Sampled For DNA

• 65 Remains ID By DNA• 75 Remains Not Identified• 1,455 Still “Missing”??

DMORT-East, Gulfport, MSAs of 20 March 2006

• 39 Recovered RemainsSampled For DNA

• 14 Remains ID By DNA• 3 Remains Not Identified

Biometrics, Automation, and Special Projects Section

Samples received: 90,133Samples processed auSTR: 80,462 Samples processed ySTR: 603 Samples processed mtDNA: 1,240

Total Comparisons Requested: 1,265

7

mtDNA Section• Environmentally Challenged• Direct References Not Available

• Cases 25 to 140 Years Old• Generally Fragmented

AFDIL Support Of The Joint POW/MIA Accounting Command

299

396426

398

399

446426

728465 482

569653

440

760

834 841

789

717

771802

450510

500

600

700

800

900

JPAC Specimens Received And Reported By AFDIL As of 31 May 2008

299 399

395

334

0

100

200

300

400

FY 97 FY 98 FY 99 FY 00 FY 01 FY 02 FY 03 FY 04 FY 05 FY 06 FY 07 FY 08Specimens Received Specimens Reported

Overall Success Rate

90

100

110

te

Signed Demineralization SOP

50

60

70

80

Jan-0

5

Mar-05

May-05

Jul-0

5

Sep-05

Nov-05

Jan-0

6

Mar-06

May-06

Jul-0

6

Sep-06

Nov-06

Jan-0

7

Mar-07

May-07

Jul-0

7

Sep-07

Nov-07

Jan-0

8

Mar-08

% S

ucce

ss R

at

90% Success Rate For Obtaining DNA Sequence Data

FY07 Accomplished802 Specimens

8

90-100% success

80-89.9% success

60-69.9% success

70-79.9% success

40 49 9% success

50-59.9% success

40-49.9% success

30-39.9% success

0-20% success

Non-demineralization success

5886 samples processed

Demineralization success

736 samples processed

Slide by S.M. Edson

Vietnam Case Example• Remains repatriated 17 boxes in 1988

• One box associated by documents to Ralph C. Bisz

I t i f l l• Interviews of locals supported the documentation

• Samples were submitted to AFDILfor DNA Analysis

1991 01A, 02A, 03A, 04A, 05A

1993 01B, 02B, 07A, 08A

2000 11A

2000 10A

Slide by J.E. O’Callaghan

2001 11B

2002 01C, 08B, 12A, 13A, 14A15A, 16A, 17A, 18A

2004 01D, 03B, 15B, 19A

The final submission was received 7/04, but was not photo-documented until 08/07, nor processed until 9/07

9

01C

15A

08B12A

17A

16A

LCDR Ralph C. Bisz, USNR

Slide by J.E. O’Callaghan

18A

01D

03B15B

Sample Quality appears to be consistent throughout all submissions

13A

Identified 21 April 2008

Resolving Extremely Commingled Skeletal Remains From the Korean War

Specimens from the Korea-208 (K208)

Slide by J.E. O’Callaghan

Slide by J.E. O’Callaghan

10

East Chosin Complex Un San ComplexChongryang-ri 50 Chongsung-ni 296Kaljon-ri 219 Okchang-ni 70Sinhung-ri 329Hwangcho-ri 22

Suan POW Camps Independent RegionsNamjong-gu 351 Midang-ri 3Okchi-ri 81 Songgwan-ri 12ggTojon-ni 4 Sinchang-ni 4Up-ri 119 Komsa-ri 18Holdong-gu 102 Sinan-ri 10Ryongpho-ri 20 Ryongsan-ri 44SMC 117 Tongju-ri 275

Kundong-ni 10

Total = 2,255 samplesSlide by J.E. O’Callaghan

Slide by J.E. O’Callaghan

Research Section•Various Grant Funded Projects•Multiple Collaborations with other Laboratories•Novel Design of Innovative Techniques•Historical and International Casework Support

Supports both the mtDNA and the nucDNA Sections

11

Utility of NC miniSTR multiplexes at AFDIL

NC miniSTR testing on K208 bones

• Bones with matching mtDNA CR sequences from three different cases (boxes) from two different camps:

Slide by R.S. Just

• When typed with the NC miniSTR multiplex, all three samples gave the same profile

Namjong-gu: Case A, Sample 06B Namjong-gu: Case B, Sample 02A Okchi-ri: Case C, Sample 04A

14,16 14,16 14,16

Utility of NC miniSTR multiplexes at AFDIL

, , ,

10,14 10,14 10,14

12,14 15,17 12,14 15,17 12,14 15,17

Slide by R.S. Just

Ancient DNA Research and the Challenge of the Punchbowl

•Korea / Kokura remains

•Powder = hardening compound

•Unable to amplify the DNA

Odile Loreille, PhD

12

osteocytelacunae

Haversian canals

Micrograph of Remains

“It is really surprising that even the osteocyte lacunae are filled with powder, because the pores that give access to them are just 200 nm in diameter”

Miranda Jans, PhD

17

118

128

107

158

140

106

97

78

80

140

147

35

57 42

Current Samples Completed

6325

147 77

118113123150

46 80

108

167 179

166185 171

101

10098

100

15 81

108107 108

182

April 8, 2008

126

108

79

105

100

9492

104

100 98

100

15 67

100

50

U.S. mtDNA DatabasesSlide by M.D. Coble

266

93

92

209

xxx

xxx

xxx

xxx

xxx

African American

Western European

Hispanic

Asian

Native American

109110

91 86

10099

98

146

148149

149

12799

34

100

9960

99Summary of Population DB SizesAfrican American 2046Asian 651W. European 2129Hispanic 2146Native American 1005

Total 7977

OGA Funding to AFDIL to Increase the mtDNA databases in these regions

Lebanon

Population STR Genotypes mtDNA HaplotypesBahrain 168 213Jordan 157 202

Dubai (UAE) 147 187

Egypt

Iraq

Bahrain

Dubai (UAE)

PakistanJordan

Egypt --- 278Iraq 146 184

Lebanon 192 195Pakistan 405 412

Total 1215 1671

Slide by M.D. Coble

13

Mini-X STRs

??

6-FAM(blue)

VIC(green)

NED(black)

60 bp 100 bp 140 bp

PET(red)

DXS7424 (63-106)

DXS6789 (118-166)

DXS7130 (97-137)

DXS7423(87-135)

GATA165B12 (82-110)

DXS101 (121-175)

160 bp 200bp

DXS9902 (160-188) *

SRY82

180 bp120 bp80 bp

DXS10147(153-169)

6-FAM(blue)

DXS6795 (89-107)

GATA172D05 (108-136)

60 bp 100 bp 140 bp 160 bp 200bp180 bp120 bp80 bp

DXS10101 (183-225)

Mini X-Plex 2

Mini X-Plex 1

•when mtDNA testing is insufficient

•no maternal reference available

•remains have most common mtDNA type

Toni Diegoli, MFS

?VIC

(green)

NED(black)

PET(red)

DXS8378 (87-115)

DXS6803(97-128)

GATA31E08 (93-133)

DXS9902 (160-188)

HPRTB(139-175)

DXS7132 (130-174)

*SRY82

mtDNA 12S Gene for Species IDBone fragments failing to yield an amplicon with Human specific primers:

Highly degraded or Non-human ?

Kimberly Sturk, MFS

Historical Cases

During the American Civil War, off the coast of South Carolina, the world’s first successful submerged war vessel, the H.L. Hunley, sank the USS Housatonic. The H.L. Hunley never made her return voyage and was presumed lost. In 1995 the wreckage of the H.L.

H.L.Hunley

USS Monitor launches from Long Island, NY in January 1862 as world's first ironclad, turreted warship. Historic action between USS Monitor and CSS Virginia in June, but sinks in a fierce storm off Cape Hatteras, North Carolina in December. Gun turret successfully recovered in August 2002 with two nearly complete skeletal remains.

was presumed lost. In 1995 the wreckage of the H.L. Hunley was discovered resting on the floor of the Atlantic Ocean.

USS Monitor

14

Historical Cases

Unknown Child from the Titanic

Czar Romanov and Family

A child was pulled from the ocean in the days that followed the ‘Titanic’ disaster and buried as an unknown.

Mozart

AFDIL’s International mtDNA Training Course

AcknowledgementsNuclear

Demris LeeTracy M. Johnson

Jeffrey HickeyDiane MuellerCarna MeyerAll Analysts

ResearchMichael Coble

Jodi IrwinRebecca Just

Kim SturkToni Diegoli

Odile LoreilleMiranda Jans

MitochondrialSuzanne Barritt-Ross

Mark WadhamsChristopher Los

Suni EdsonCarla Paintner

Jackie Raskin-BurnsSarah Bettinger

AutomationTed Anderson

Tracey JohnsonColin StevenAll Analysts

All Technicians

Training & EducationFaith Patterson

Richon Tate

All TechniciansMiranda Jans

Melissa ScheibleJessica Saunier

AFDIL Senior StaffDr. Louis Finelli

Mr. James Canik jenney.ocallaghan@us.army.milThe opinions and assertions contained herein are solely those of the author and are not to be construed as official or as views of the National Institutes of Justice, the U.S. Department of Defense, or the U.S.

Department of the Army.

Sarah BettingerJennie McMahon

Chad ErnstAll Analysts

All TechniciansOther

AFDIL Administrative SupportQuality Control Department

Evidence Custodians

15