3 Fully automated, Field forward Rapid DNA Typing for Military, Intelligence, and Law Enforcement Applications090413
4 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 processingNo reagent loading—reagents should be incorporated into the single-use consumableNo instrument calibration or assemblyNo data interpretation—automated allele calling and searchingReagent Stability/Out-of-the-box functionality. The System will be placed in a field forward environment.No temperature, humidity, or particulate controlNo refrigerationNo tools or technicians to install/align systemGenerator power
5 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 trackingEncryption of all generated data and databaseFlexibility. The system must provide a customizable foundation for ﬁeld forward forensic DNA analysis.Touch sample analysisKinship analysisMulti-assay capabilityFor first bullet: As discussed in the previous talk, the system must be ruggedized
6 ANDE System Components and Process Flow Collection SwabFully AutomatedInstrumentBioChipSet™ CassetteAutomatedData Analysis6
7 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.
18 STR Analysis, How Does It Work? Unknown: Evidence Sample:Blood, Body FluidsCigarette ButtHandled Objects (cup, pop can, water bottle, etc)Murder WeaponKnown: Reference Sample:Convicted OffendersArresteesSuspectsPaternityDNA PurificationDNA PurificationDNA QuantitationDNA QuantitationSTR AmplificationCE Separation & DetectionDNA Profile GenerationDatabaseSearch/Match
19 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 AATG7 repeats (maternal)8 repeats (paternal)AATG~
20 STR Analysis, How Does It Work? Unknown: Evidence Sample:Blood, Body FluidsCigarette ButtCupMurder WeaponKnown: Reference Sample:Convicted OffendersArresteesSuspectsPaternityDNA PurificationDNA QuantitationSTR AmplificationCE Separation & DetectionDNA Profile GenerationDatabaseSearch/MatchSTR Amplification
22 DNA Profiling is Extraordinarily Accurate LocusAlleleValue1 inCombinedD5S818110.2533130.21529.179VWA170.28158.8781FGA250.1854260.218512.351005D8S1179140.165616.2916,364D21S11280.1589310.278211.31185,073D18S510.13740.139126.184,845,217D3S1358150.3841160.14079.2544,818,259D13S317100.33940.048030.691.38 x 109D7S8200.177231.854.38 x 1010D16S5390.11260.321213.806.05 x 1011THO160.23180.281418.621.13 x 1013TPOX80.53483.503.94 x 1013CSF1PO0.21691221.288.37 x 1014Regions 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 matchJ. Bulter,The probability of a random match for this STR profile in the U.S. population is less than 1 in 837 trillion
23 Traditional DNA Forensic Laboratory Infrastructure Capital EquipmentLab Supplies and ReagentsComputers and softwareLaboratory TechniciansForensic AnalystsSample Prep LabAmplification LabSeparation & Detection LabMost 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 ftDNA Unit ~$56 M, 70,000 sq ft, annual operational budget >$2 MForensic Analysis
24 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
25 ANDE: NetBio’s Rapid DNA Instrument Instrument Weight: 50 kg (two person lift)Instrument Size: 68 x 42 x 59 cmRuggedized to MIL STD 810F for shock / vibrationNo calibration or realignment required after transportRFID ReaderTouchscreen MonitorBioChipSet Access DoorFolding Carry HandleGPS Window
26 Field Forward Testing to MIL STD 810F The ANDE System has under gone extensive ruggedization and field testingShockBench HandlingRestrained Cargo VibrationFragilityOperability with Generator Power
27 Process Flow: DNA Purification Insert Swab (x5)Cell LysisDNA PurificationElute DNAAll BioChipSet process steps are controlled using pneumatic pressures.Guanidinium/Silica filter based purification.Self-normalizing to delivery consistent range of DNA concentration for amplification.
28 Process Flow: Rapid Multiplexed STR Amplification Purified DNA SampleReconstitute PCR CakeTransfer to PCR ChambersAmplificationPCR 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.
29 Process Flow: Electrophoretic Separation and Detection Join PCR Product & FormamideReconstitute 5 ILS Cake/1 ALInjection 5 Samples/1 ALElectrophoretic SeparationFluorescence Detection
30 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 andNo data review or analysis by a forensic analyst is required.
31 ANDE TestingU.S. Testing Partners: USACIL, FBI, and NIST have been performing laboratory testing for the past 12 monthsU.S. Military/Intelligence Exercise Trident Specter: deployed in maritime operationsSOCOM: initial laboratory testing performed, currently deployed for field testingAustralia: Successfully deployed in field exercise Q3 2013DHS: Immigration pilot scheduled for Q4 2013
32 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 OperationsDatabase EnrollmentCounter Terrorism/Counter NarcoticsMaritime OperationsEnemy Combatant IdentificationCasualty identificationHomeland SecurityImmigration processingMass Disaster Victim IdentificationBorders and ports protectionPassport/document fraudHuman trafficking preventionLaw EnforcementArrestee processingCrime laboratory supportHighlight both kinship applications and linking people to crimes