DNA Analysis In The Identification Process

Timothy P McMahon PhD Director, DoD DNA Registry

Armed Forces Medical Examiner System

National League of POW-MIA Families Arlington, VA. June 23, 2017

Purpose

-AFMES / DoD DNA Registry (AFDIL)

- DNA 101

- Way ahead

- Where you fit in

DNA FAQ

http://www.dpaa.mil/

AFMES Missions Supported By AFDIL

Present Day Accounting Past Accounting

FRS Databasing World Wide Support

www.bbc.co.uk

https://www.google.co.il/search?q=images+of+human+bones+exposed+to+the+environment&biw=1584&bih=719&tbm=isch&tbs=simg:CAQSEgm1oUy70AjM2iF0yJK9BI3cHQ

Laboratory Accreditations & Standards

American Society of Crime Laboratory Directors-Laboratory Accreditation Board (ASCLD – ISO 17025 International Certification)

Federal Bureau Of Investigation (FBI) Quality Assurance Standards

DoD DA Oversight Committee (Defense Science Board 1995)

The Human DNA Genome

Nuclear DNA ~3.2 billion base pairs

(bp)

Mitochondrial DNA

16,549 bp

23 Pairs of Chromosomes

The Nuclear Genome contains approximately

25,000 Genes

Chromosome #23

Female

1 4 5 6

9 10 11 12

13 14 15 16 17

19 20 21 22

7 8

18

23 XX

or

XY

Male

10,000X magnification of X and Y chromosomes

3 2

Ability to ask Directions (DIR)

Nuclear DNA Shared Between Relatives

9 Amount of total nuclear DNA in common with ones relatives

Comparison of DNA Testing Methods

Nuclear DNA High discrimination

Two copies/cell

Best reference are mother, father, siblings &/or offspring

•Several years

•Quick turn-around

Mitochondrial DNA Limited discrimination

Multiple copies/cell

References from any maternal relative

Decades to centuries

Slower turn-around

Y- DNA Limited discrimination

Only one copy/cell; from the father

References from any paternal relative

Several years

Quick turn-around

Past Accounting Process

• DPAA-Lab samples are processed on a rolling basis – 700+ skeletal samples in progress at any one time

– Samples are reprioritized on a routine basis

• DPAA scientist in charge of prioritizing

• AFDIL sends Data summary to DPAA

• DPAA request Comparison and BTB reports

• Average turn around time (TAT): – Extraction to Report ~55-85 Mission Days

DNA Tests Reported By AFDIL As Of May 31, 2017

93% Success Rate Obtaining MtDNA Sequence Data 40% Success Rate Obtaining Au-STR DNA Data 49% Success Rate Obtaining Y-STR DNA Data 42% Success Rate Obtaining NGS Data

0200400600800

100012001400160018002000220024002600280030003200

FY 09 FY 10 FY 11 FY 12 FY 13 FY 14 FY 15 FY 16 FY 17

MtDNA Analyses Autosomal DNA Analyses YSTR Analyses

NGS Analyses Alternate-Direct References

DNA Reports May 31, 2017

FY Believe to Be Reports Addendum

Reports Total Reports

2011 53 88 141

2012 60 160 220

2013 55 159 214

2014 75 108 183

2015 71 109 180

2016 133 71 204

2017 101 65 166

Notable Casework Advances

1992: First Use of MtDNA for Past Accounting Casework

1998: First ASCLD-LAB Accredited Laboratory for MtDNA Testing

2006: Demineralization Buffer 2010: 12S rRNA: Human vs Non-Human 2013: Low Copy Number Y-STR testing 2014: Improved DNA Purification 2015: New 23 locus auSTR Kit 2016: NGS mtDNA Capture Protocol for chemically modified samples

90-100% success

80-89.9% success

60-69.9% success

70-79.9% success

40-49.9% success

50-59.9% success

30-39.9% success

0-20% success

2006:Demineralization Success

Pre-Demineralization Post- Demineralization

2014: Improved DNA Purification

• FY13: Old Purification Process – Y-STR: 24% – auSTR: 26% – mtDNA Sequencing: 90%

• FY14 -15: Improved DNA Purification Process – LCN-Y: 48% – auSTR: 48% – mtDNA Sequencing: 92%

• FY16 Success Rates – LCN-Y: 47% – auSTR: 48% – mtDNA Sequencing: 92%

2015: New Autosomal STR Kit

• MiniFiler: 8 loci less than 215 bp

• Eleven loci less than 250 bp

• Inclusion of 11 mini-STRs and 12 larger loci provides enhanced performance with degraded DNA

2016: NGS mtDNA Capture Method

• Kokura Mortuary • Chemically Modified

– 40-50% formaldehyde/ Bad for DNA

• 16 year project by AFDIL

DNA/Protein Fixation Issue

http://www.precisionnutrition.com/wordpress/wp-content/uploads/2009/12/Figure-1-Histones-1024x1022.jpg

Illumina MiSeq

2016 NGS mtDNA Capture Protocol

• Samples processed in duplicate – Slow process, 2 weeks per sample

– 3 to 5 samples per month

• First report issued March 17, 2016 – 250 samples have been reported to DPAA

• 28 BTB reports: 2 for SEA

– ~10 Analysis per month

• Optimize and increasing through-put – Increase instrument and personnel

Extraction

DNA Repair

Library Preparation

Library PCR

Hybridization Capture

Capture PCR

Sequencing and Data analysis

Casework Automation

• Casework Manual Method – 72 samples processed in 12 hours

– Multiple scientist needed

• Automated Family Reference Specimen processing – 1 scientist needed to run robot

• Casework HTP Method – USS OK, USS WV, etc

– 72 samples processed in 12 hours

– 1 scientist needed to run robot

• Frees scientist to work more difficult cases

Current Problems and Gaps

Environmental Influences • DNA Damage

FFPE Repaired Unrepaired

Same mitotype and no auSTR or YSTR results

• Whole mtDNA genome sequencing – Next Generation

Sequencing protocol

– Works in conjunction with NGS mtDNA Capture method

– Individuals with common mtDNA distinguishable

– Implemented October 2016

No mtDNA, YSTR, or auSTR References

• Single Nucleotide Polymorphism Capture method – Millions of identity and kinship single nucleotide

polymorphisms (SNPS) across nuclear genome • Ancestry.com and 23AndMe.

– Working with Parabon in conjunction on DoD funded research to develop software to work with DPAA samples

– Identified 10,000 SNPS that can identify unrelated from related out to a 4th degree relative ( second cousin..)

– Opens up reference pool and removes the need to do auSTR and YSTR testing as well as having a deep reference pool

No mtDNA, YSTR, or auSTR References

Family Reference Samples

• YOU Are The Key To The Identification Process • Family References Are Collected Under Informed Consent With the Donor

and Can Only Be Used by AFMES-AFDIL For Human Remains Identification • All FRS Samples Are Treated As a Medical Specimen • Protected Under the Health Insurance Portability and Accountability Act of

1996 (HIPAA) For Personally Identifiable Information (PII) To Release of Information

• FRS Database Information Is Restricted and Not Shared Or Uploaded To Any Outside Agency

• Release of Any HIPAA Information With PII Must Be With Consent of Donor

How Does This Help You and DPAA

• Reduced time spent processing samples • Reduced reagent costs due to increased

success rates – Allowed for hiring 24 new staff 2 years ahead of plan

• Increased number of samples reported to DPAA – 2014: 1,300 samples reported

– 2015: 1,700 samples reported

– 2016: 2,100 goal for FY16; 3,142 FY16

• Authorized 15 new scientists – 39 new scientists

Unclassified

Director DoD DNA Registry: Timothy.P.McMahon10.civ@mail.mil

Slide Number 1PurposeDNA FAQSlide Number 4Laboratory Accreditations & StandardsThe Human DNA Genome Slide Number 7Slide Number 8Nuclear DNA Shared Between RelativesSlide Number 10Past Accounting ProcessDNA Tests Reported By AFDIL�As Of May 31, 2017 DNA Reports�May 31, 2017Notable Casework AdvancesSlide Number 15 2014: Improved DNA �Purification�2015: New Autosomal STR Kit�2016: NGS mtDNA Capture MethodSlide Number 192016 NGS mtDNA Capture ProtocolCasework AutomationSlide Number 22Environmental InfluencesSame mitotype and no auSTR or YSTR resultsNo mtDNA, YSTR, or auSTR ReferencesNo mtDNA, YSTR, or auSTR ReferencesFamily Reference SamplesHow Does This Help You�and DPAA Slide Number 29