NetBio - Vice President – Strategic Planning Melissa May NetBio - Vice President – Strategic Planning Date: 22/10/2013

Heading

Fully automated, Field forward Rapid DNA Typing for Military, Intelligence, and Law Enforcement Applications 090413

Requirements: ANDE Rapid DNA Analysis System (I) In 2009 NetBio was awarded the ANDE contract, a competitive R&D program funded by a consortium of 5 Federal agencies to develop a Rapid DNA Analysis System based on: Ease-of-use. The Operator has no technical training. No manual processing No reagent loading—reagents should be incorporated into the single-use consumable No instrument calibration or assembly No data interpretation—automated allele calling and searching Reagent Stability/Out-of-the-box functionality. The System will be placed in a field forward environment. No temperature, humidity, or particulate control No refrigeration No tools or technicians to install/align system Generator power

Requirements: ANDE Rapid DNA Analysis System (II) Ruggedization. The system must be hardened for transport to and operation in deployed environments. Rapid. DNA analysis must provide results within a time period that allows decisions to be made BEFORE releasing a person of interest. Data security. All aspects of system operation and data generation must be secure. User and sample tracking Encryption of all generated data and database Flexibility. The system must provide a customizable foundation for field forward forensic DNA analysis. Touch sample analysis Kinship analysis Multi-assay capability For first bullet: As discussed in the previous talk, the system must be ruggedized

ANDE System Components and Process Flow Collection Swab Fully Automated Instrument BioChipSet™ Cassette Automated Data Analysis 6

ANDE System Specifications Sample Types: Fresh and dried buccal and blood swabs, purified DNA. Number of Samples Per Run: 5 plus allelic ladder. Analysis Time: DNA profile in 84 minutes. Operator Responsibility: Insert samples and initiate run. No other manipulations are required. Reagent Stability: Reagents are stable for 6 months at room temperature, no refrigeration required. Data Analysis: Fully automatic, integrated expert system software. No analyst review required. Sample Tracking: Integrated RFID scanner on board the instrument allows tracking of RFID enables sample swabs. System Maintenance: Field serviceable, annual maintenance. Data Security: Password protected access and data encryption.

Operating the ANDE System: Collect Samples

Enter Sample ID

Scan a Sample with the Integrated RFID Reader

Insert Sample and Press the Done Button

Load the Sample into the BioChipSet Repeat this 5x

Press the Done Button

Insert the BioChipSet Cassette Point of scanning is sample tracking within the chip

Initiating the Run

DNA Processing Complete

Female DNA Profile

STR Analysis, How Does It Work? Unknown: Evidence Sample: Blood, Body Fluids Cigarette Butt Handled Objects (cup, pop can, water bottle, etc) Murder Weapon Known: Reference Sample: Convicted Offenders Arrestees Suspects Paternity DNA Purification DNA Purification DNA Quantitation DNA Quantitation STR Amplification CE Separation & Detection DNA Profile Generation Database Search/Match

STR Amplification: What Are Short Tandem Repeats (STRs)? Human DNA is 99% identical from person to person. However, some regions of the genome vary, and one type of variable region is the Short Tandem Repeat (STR). Short Tandem Repeats (STR) are repetitive sequences: Tetranucleotide: AATG AATG AATG AATG 7 repeats (maternal) 8 repeats (paternal) AATG ~

STR Analysis, How Does It Work? Unknown: Evidence Sample: Blood, Body Fluids Cigarette Butt Cup Murder Weapon Known: Reference Sample: Convicted Offenders Arrestees Suspects Paternity DNA Purification DNA Quantitation STR Amplification CE Separation & Detection DNA Profile Generation Database Search/Match STR Amplification

Female DNA Profile

DNA Profiling is Extraordinarily Accurate Locus Allele Value 1 in Combined D5S818 11 0.2533 13 0.2152 9.17 9 VWA 17 0.2815 8.87 81 FGA 25 0.1854 26 0.2185 12.35 1005 D8S1179 14 0.1656 16.29 16,364 D21S11 28 0.1589 31 0.2782 11.31 185,073 D18S51 0.1374 0.1391 26.18 4,845,217 D3S1358 15 0.3841 16 0.1407 9.25 44,818,259 D13S317 10 0.3394 0.0480 30.69 1.38 x 109 D7S820 0.1772 31.85 4.38 x 1010 D16S539 0.1126 0.3212 13.80 6.05 x 1011 THO1 6 0.2318 0.2814 18.62 1.13 x 1013 TPOX 8 0.5348 3.50 3.94 x 1013 CSF1PO 0.2169 12 21.28 8.37 x 1014 Regions of chromosomes that are looked at have no phenotypical information-except for gender. This table looks complicated, but makes the point that as you interogate more regions of the genome the probability of a random match J. Bulter, http://www.cstl.nist.gov/biotech/strbase/fbicore.htm The probability of a random match for this STR profile in the U.S. population is less than 1 in 837 trillion

Traditional DNA Forensic Laboratory Infrastructure Capital Equipment Lab Supplies and Reagents Computers and software Laboratory Technicians Forensic Analysts Sample Prep Lab Amplification Lab Separation & Detection Lab Most Foresnic Analysts have a masters degree and require 6 months of training, continuing education in a forensic lab before reaching full staff. Washing dishes, and autoclaving for sterilization, U.S. Crime Lab $210 M, 351,000 sq ft DNA Unit ~$56 M, 70,000 sq ft, annual operational budget >$2 M Forensic Analysis

Five Lab Processes in One Portable Device Rapid DNA Analysis for Human Identification is the automated generation of STR profiles (DNA fingerprints) by non-technical operators outside the laboratory. Rapid DNA Analysis for Human Identification is the automated generation of STR profiles (DNA fingerprints) by non-technical operators outside the laboratory. Human Analyst Profile Match Hours - Weeks

ANDE: NetBio’s Rapid DNA Instrument Instrument Weight: 50 kg (two person lift) Instrument Size: 68 x 42 x 59 cm Ruggedized to MIL STD 810F for shock / vibration No calibration or realignment required after transport RFID Reader Touchscreen Monitor BioChipSet Access Door Folding Carry Handle GPS Window

Field Forward Testing to MIL STD 810F The ANDE System has under gone extensive ruggedization and field testing Shock Bench Handling Restrained Cargo Vibration Fragility Operability with Generator Power

Process Flow: DNA Purification Insert Swab (x5) Cell Lysis DNA Purification Elute DNA All BioChipSet process steps are controlled using pneumatic pressures. Guanidinium/Silica filter based purification. Self-normalizing to delivery consistent range of DNA concentration for amplification.

Process Flow: Rapid Multiplexed STR Amplification Purified DNA Sample Reconstitute PCR Cake Transfer to PCR Chambers Amplification PCR uses Promega PowerPlex® 16 System. PCR reagents have been lyophilized for long-term stability at room temperature (>2 years). Rapid amplification: 31 cycles, 19.5 min.

Process Flow: Electrophoretic Separation and Detection Join PCR Product & Formamide Reconstitute 5 ILS Cake/1 AL Injection 5 Samples/1 AL Electrophoretic Separation Fluorescence Detection

Data Analysis and DNA Profile Generation Expert System software performs automated data processing & allele designation. Three data output files are automatically generated for database searching and No data review or analysis by a forensic analyst is required.

ANDE Testing U.S. Testing Partners: USACIL, FBI, and NIST have been performing laboratory testing for the past 12 months U.S. Military/Intelligence Exercise Trident Specter: deployed in maritime operations SOCOM: initial laboratory testing performed, currently deployed for field testing Australia: Successfully deployed in field exercise Q3 2013 DHS: Immigration pilot scheduled for Q4 2013

Rapid DNA Analysis: An Enabling Technology Rapid DNA Analysis for Human Identification is the automated generation of STR profiles (DNA fingerprints) by non-technical operators outside the laboratory. Major applications to enhance societal safety include: Military and Intelligence Operations Database Enrollment Counter Terrorism/Counter Narcotics Maritime Operations Enemy Combatant Identification Casualty identification Homeland Security Immigration processing Mass Disaster Victim Identification Borders and ports protection Passport/document fraud Human trafficking prevention Law Enforcement Arrestee processing Crime laboratory support Highlight both kinship applications and linking people to crimes

830 Winter Street, Waltham, MA 02451 www.netbio.com Melissa.May@netbio.com 781-981-8273