Rapid DNA Analysis DNA: the Ultimate Biometric? Advances in Molecular Processing and Analysis

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Rapid DNA Analysis

DNA: the Ultimate Biometric?

Advances in Molecular Processing and

Analysis

Presentation at

NSF Workshop on Fundamental Research Challenges for

Trustworthy Biometrics

Dr. Joan Bienvenue

Chief Scientist, Program Manager, Lockheed Martin

and

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OUTLINE

■

Future Directions for Biometrics

▪ DNA Microfluidics

• Where are we at? Where do we need to be?

■

Beyond STRs: Next Generation

Technological Advances

▪ SNPs

▪ NextGen sequencing

■

New Technology, New Problems?

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Biometric Modalities

(4)

Challenges with DNA As A Biometric

• Different applications,

different specs

• Portability

• Training of end users

• LCN

• Casework

• Evidence quality

• Interpretation of

results

• Speed of analysis

• Court acceptance

(5)

Our Motivation

9 hrs, 50 min (average) 8 hrs, 15 min (rush) Detection PCR Amplification Dilution gDNA Quantification Cell Lysis/ DNA Extraction

Qiagen EZ1 ABI 7000

RT-PCR Tecan Genesis ABI 9700 Multimek ABI 3130 1hr 45 min 2 hr 20 min 15 min 3 hr 30 min 2 hr

Current Approach

LM-ZyGEM/MicroLab Approach

INTEGRATED DEVICE

Step 1: Sample Collection Step 2: Liquid Extraction Step 3: Polymerase Chain

Reaction Step 4: Electrophoresis

Microfluidics

Approach

•Inexpensive

•Portable

•Robust

Alpha Device

(6)

Integrated Instrument:



Sample in-answer out



Turn-key operation



Miniaturizable footprint



Portable



Automated



Unskilled personnel

RapI.D. STR Chip – Micro-Analysis

Microvolume Analysis:



Ultrafast processing



Open channel architecture



Reduced reagent needs



Closed analysis environment



Small analytical footprint

(7)

Anticipated LM-ZM Instrument

• Ruggedized instrument at

TRL 6+

• Modular Component design

to support maintainability

• Easily portable

• Removable PC

• 4, 8, 12, 16 channel

cartridges containing

reagents

• “Plug and Play” concept

• Portable (< 40 lbs)

(8)

Micro-Total Analysis STR Profiling in <45 min?

STR Profile

DNA Purification DNA Amplification <4 min Injection Separation/D etection 8 min <2 min <20 min

SPE Inlet

Extraction Amplification Separation Injection

+5 min for fluidic pumping, etc.



Multiplex amplification



High resolution separation

(9)

Rapid Microfluidic DNA Analysis – A Reality

• Multiple competitors,

research born out of high

quality university effort

• Close ties to biometric and

forensic analysis

communities

• Small companies working

together with universities,

larger corporations to bring

the best in technology and

innovation to these analyses

• Rapid, portable analysis is

possible

(10)

Expedite the 3 Major Sample Preparation/ Analysis Steps:

•Solid Phase Extraction (SPE)

•Polymerase Chain Reaction (PCR)

•Separation and Detection

Motivation – Expedite and Miniaturize

9 hrs, 50 min (average) 8 hrs, 15 min (rush) Detection PCR Amplification Dilution gDNA Quantification Cell Lysis/ DNA Extraction

Qiagen EZ1 ABI 7000

(11)

Expedite the 3 Major Sample Preparation/Analysis Steps:

•Solid Phase Extraction (SPE)

•Separation and Detection

Motivation – Expedite and Miniaturize

Qiagen EZ1 ABI 7000

(12)

Generation of High Quality, PCR-Ready DNA ….

4 min 8 min 2 min 40 min

SPE Inlet

Extraction Amplification Separation Injection DNA Prep 0 2 4 6 8 10 12 14 16 18 20min



Time reduced from 20 min to <4 min



Volume reduced from several hundred



L



A simple one-step process performed

(13)

Expedite the 3 Major Sample Preparation/Analysis Steps:

•Polymerase Chain Reaction (PCR) •Separation and Detection

Motivation – Expedite and Miniaturize

Qiagen EZ1 ABI 7000

RT-PCR Tecan Genesis

ABI 9700

ABI 3130 Multimek

(14)

Rapid Amplification of STR Loci ….

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Time (min)

Conventional PCR: IdentiFiler®

IdentiFiler® amplification: 1 ng template DNA 25 µL reaction volume 90 70 50

T

emperature

40 80 120 160 200

Total amp. time:

~210 min

11 mininitial denature, 95 ºC 32 cycles

60 sec@ 94ºC; 60 sec@ 59ºC;

60 sec@ 72ºC

(16)

Microchip IR-PCR Amplification: IdentiFiler®

2 mininitial denature, 95 ºC 32 cycles

5 sec@ 94ºC; 10 sec@ 59ºC; 10 sec

@ 72ºC

1 minfinal extension 72 ºC IdentiFiler® amplification: ~1 ng template DNA 0.5 microliter volume 90 70 50

T

emperature

Total amp. time:

~26 min

Time (min)

5 10 15 20 25

Modified conditions from Vallone et. al.FSI: Genetics,2008, 3, 42-45

(17)

Expedite the 3 Major Sample Preparation/Analysis Steps:

•Separation and Detection

Motivation – Expedite and Miniaturize

Qiagen EZ1 ABI 7000

ABI 9700

ABI 3130 Multimek

(18)

Peak Resolution in Short Channels

D3S1358 and THO1 _9.3/10



High resolution separations

in short distances (L

_eff

)

allows for reduced chip

footprint and reduced

separation times



9.3/10 resolved in 7cm L

_eff

(19)

Expedite the 3 Major Sample Preparation Steps:

•Solid Phase Extraction (SPE)

•Polymerase Chain Reaction (PCR) •Separation and Detection

Motivation – Expedite and Miniaturize

Qiagen EZ1 ABI 7000

ABI 9700

ABI 3130 Multimek

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Portable DNA Analysis

• Easy, less than 1 hr of

training to use

• Automated allele

calling/expert system

software

• Automated profile

generation

• Automated database

searches

• How does this impact the

quality, reliability, and

acceptability of results?

(23)

OUTLINE

■

Future Directions for Biometrics

▪ DNA Microfluidics

• Where are we at? Where do we need to be?

■

Beyond STRs: Next Generation

Technological Advances

▪ SNPs

▪ NextGen sequencing

(24)

• Useful in cases where a STR profile

doesn’t match an existing profile in

CODIS or an identified suspect

• Provide information useful to the

investigation improving the ability to

identify potential suspects

SNPs for Ancestry & Phenotype Inference

• Will not directly identify a single suspect individual but

will:

• assist in prioritizing suspect processing

• corroborate witness testimony

(25)

Forensic SNPs

■

Individual Identification SNPs (IISNPs)

■

Lineage Informative SNPs (LISNPs)

■

Ancestry Informative SNPs (AISNPs)

■

Phenotype Informative SNPs

(PISNPs)

SNPs that

collectively give a

high probability of an

individual’s ancestry

being from one part

of the world or being

derived from two or

more areas of the

world.

SNPs that

collectively give a

high probability of an

individual’s ancestry

being from one part

of the world or being

derived from two or

more areas of the

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Ancestry Informative ISNPs -

Duffy Null Allele

• Duffy blood group identifies phenotypes associated with two

proteins that appear on the surface of red-blood cells

• Plays an important role in susceptibility to malaria infection

• The Fy(a-b-) phenotype (i.e., not expressing the receptor)

represents an adaptation to living in malaria-endemic regions

• Predominant in African populations esp. those from West Africa.

(27)

Forensic SNPs

■

Individual Identification SNPs (IISNPs)

■

Lineage Informative SNPs (LISNPs)

■

Ancestry Informative SNPs (AISNPs)

■

Phenotype Informative SNPs

(PISNPs)

SNPs that

collectively give a

high probability Of

a phenotypic

marker of a

specific physical

trait.

SNPs that

collectively give a

high probability Of

a phenotypic

marker of a

specific physical

trait.

(28)

G. Tully, Forensic Science International: Genetics 1, 105 (2007

Phenotype Informative SNPs

HERC 2

http://voiland.org/blog/wp-content/uploads/2008/02/blue.jpg

• Five genes (

SLC24A, ASIP,

MC1R, SLC45A2,

OCA2

)

involved in pigment expression

• HERC 2 (part of OCA2) has

SNPs strongly associated with

blue eyes

(29)

• Rapid sequencing

of the entire

genome of an

individual

• Currently cost is

$30-100K

• NIH searching for

the technology for

the $100 genome

(30)

• Technologies are

evolving rapidly for

full genome seq

(FGS)

• None capable of

doing this in a

cost-effective way

• PacBio claims FGS

15 min for $100 by

2015

(31)

OUTLINE

■

Future Directions for Biometrics

▪ DNA Microfluidics

• Where are we at? Where do we need to be?

■

Beyond STRs: Next Generation

Technological Advances

▪ SNPs

▪ NextGen sequencing

(32)

• Taking DNA into the field, how do we maintain the quality

and reliability of results?

-

What will the controls be?

-

How will instrument performance be monitored?

-

Validation of instrument, methods, chemistry?

-

Training and certification of users?

-

Audits, reviews, quality control measures?

• Automated data analysis, what happens when something

goes wrong or the results are uninterpretable/need expert

analysis?

-

Will data be shared with a laboratory for analysis?

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• What weight will the field results carry?

-

Will samples need re-analysis in the laboratory for court

acceptance?

-

Can results be used to arrest, to hold, to convict?

-

State vs Federal vs Global, different standards, different access,

different collection requirements?

• What policy has to change/be adapted to permit samples to

be run in the field, in a booking station, at a checkpoint?

-

Can the data generated be uploaded to databases?

-

Can the data generated be used to search databases?

-

What samples are uploaded to databases?

(34)

• NextGen racial profiling?

-

How and when do we use phenotypic

genetic information?

-

Does ancestral/phenotypic testing violate

civil rights?

• What other information do we get when we

sequence and how do we prevent abuse?

-

Medical conditions?

-

What if genes are found that can be

associated with a pre-disposition to

violence? Murder? Sexual Assault?

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