Presently
9/9/2014
Richard A. Guerrieri Research Leader – Human Identity Battelle [email protected]
Next Generation Sequencing – Quality Assurance Considerations for the Forensic DNA Laboratory International Symposium on Human Identification Phoenix, AZ
October 2, 2014
1
Outline • Forensic DNA – where we’ve been • Next-Generation Sequencing - what does it introduce • Quality Assurance – how to navigate going forward
2
TWGDAM • Technical Working Group for DNA Analysis Methodologies
• Formed by the FBI in 1988 (now SWGDAM) • Twelve laboratories across North America
1
9/9/2014
Early Mission of TWGDAM • Early Discussion Topics RFLP
PCR
− Enzyme
DQA1, Polymarker
− Gel Lengths
D1S80 (Horizontal, Vertical Gels)
− Ethidium Bromide
STRs (Silver Stain, Fluorescence)
• How to Approach Validation – TWGDAM Guidelines − Reproducibility & Concordance − Sensitivity & Degradation − Non-Probative Samples / Mixtures
Early Mission of TWGDAM • Discussion and Collaboration Identified an objective to achieve compatibility between
laboratories
Recognized it would be necessary to establish common
controls and practices
The goal became the capability to share data
− National DNA Index System (NDIS)
DNA Identification Act of 1994 • A statute passed by Congress related to DNA testing: Authorized the formation of the DNA Advisory Board
− Goal to evaluate current technology and quality practices for forensic DNA and database laboratories − Responsibility to recommend quality assurance practices to the FBI Director for issuance as standards for DNA testing laboratories
• DNA Quality Assurance Standards formally issued to the forensic community − Forensic DNA Testing Laboratories: October 1, 1998 − Convicted Offender DNA Testing Laboratories: April 1, 1999
2
9/9/2014
DNA Quality Assurance Standards • Covers a wide range of quality affecting practices − Personnel, Facilities, Evidence Handling, Validation, Analytical Procedures, Equipment Maintenance/Calibration, Report Writing, Data Review, Audits, Proficiency Testing, Corrective Action
• Who is required to comply with the DNA QAS? − The FBI Laboratory − All laboratories that receive federal funds pursuant to the Act − Laboratories that participate in the NDIS
DNA QAS and Next-Generation Sequencing • Does next-generation sequencing (NGS) introduce any new requirements?
• Would all existing requirements directly apply? • Are the current DNA QAS relevant to NGS forensic and database applications?
8
CE
DNA Quant
Normalize
PrePCR
PCR Amp
DNA Quant
Normalize
PrePCR
PCR Amp
Inject (CE)
Interpret
NGS
Library Construction
Sequence
Interpret
9
3
9/9/2014
CE
DNA Quant
NGS
Normalize
PrePCR
PCR Amp
QA - Most Are Compatible
DNA Quant
Normalize
PrePCR
PCR Amp
Inject (CE)
Interpret
QA – Other Steps Are New
Library Construction
Sequence
Interpret
10
NGS: Key Areas to Consider • Library Construction − Controls / Quality Measures − Instrumentation / Performance Acceptance − Facilities / Work Flow
• Sequencing − Controls / Quality Measures − Reagents − Pooling / Multiplexing
• Interpretation − Thresholds − Bioinformatics − Data Basing / Expert Systems 11
How Do We Address The DNA QAS For NGS? • Standard 2 Definitions: Contamination – Unintentional introduction of exogenous DNA into
a DNA sample or PCR − Do these terms capture Library Materials?
Critical Reagents – Empirical studies required for calibration or
performance checks required prior to use − How do you conduct performance checks on sequencing reagents/instrumentation?
12
4
9/9/2014
Standard 2: Definitions • Methodology – Analytical processes/procedures used to support a DNA typing technology Extraction methods, quantitation methods
− Library Construction Methods
• Technology – The type of DNA analysis performed STR technology; Mitochondrial Sequencing Technology
− May Need NGS Term(s): Example - SNP Typing
13
Standard 2: Definitions • Positive Amplification Control – used to determine if the PCR performed properly (introduced at amplification) − Are multiple controls required? - Do you need a step to trouble-shoot the library preparation?
• Negative Amplification Control – used to detect DNA contamination of the amplification reagents − What is comparable for NGS – is a negative control required for each step (library preparation, PCR amplification, sequencing)? - What do you learn from the control at each step?
• Reagent Blank Control – used to monitor for contamination from extraction to final analysis − Is this still applicable?
14
New Terms to Consider • De-Multiplex • Index Tags / Indexing • Library • Sequencer • Others 15
5
9/9/2014
Standard 6: Facilities • Is the laboratory designed to minimize contamination? Laboratories must demonstrate a separation in time or space
for examinations, extractions and PCR setups - Amplified DNA must be generated (thermal cyclers), processed and maintained in room(s) separate from the examination, extraction and PCR setup areas.
For robotics, accommodations for analysis through
amplification in a single room, if supported by internal laboratory validation - If contamination occurs, it is minimized, addressed and less than or equal to that observed when procedure is performed manually in separate rooms.
Standard 6: Facilities • Completely separate work areas? Pre-PCR Work Post PCR and Pre-Library Work Post Library Amplification and Sequencing
• Combination of separate work areas with dedicated one-way work flow?
Standard 9: Analytical Procedures The laboratory must identify all critical reagents and detail the
QC measures in place − Pass/Fail criteria (range of acceptable results) − Procedures for acting upon unacceptable data − Documentation of the approval/rejection of data
Critical reagents and critical instrumentation require
acceptance testing prior to use on forensic/database samples
6
9/9/2014
Standard 9: Analytical Procedures So what might be an NGS critical reagent or critical instrument?
− PCR Reaction/Enzyme Mixes − Primer Sets − Purification & Normalization Beads − Thermal Cycler − Sequencing Kits
How do we define acceptance testing for NGS prior to actual
use on forensic/database samples?
Standard 9: Analytical Procedures • Does the laboratory have and follow written general guidelines for the interpretation of data? Laboratories must monitor analytical procedures using
appropriate controls and standards
This includes requiring an assessment of controls for defined
results (blanks, negative controls); correct allele types for positive controls − Assessment must be documented
Standard 9: Analytical Procedures • What might be appropriate controls and standards for NGS to monitor the analytical process?
• What would constitute defined acceptable results? Extraction reagent blanks
− Not applicable PCR no template controls
− Introduced during library preparation Positive Controls
− Introduced during library preparation
21
7
9/9/2014
Standard 9: Analytical Procedures • Other areas of the analytical method that may be important to consider monitoring? Multiplexing
− Index Tags
Interpretation
− Thresholds - Analytical and/or Stochastic - One base line for delineating noise and defining alleles? - Acceptance of increased data (+/- stutter); sequence variants
22
Standard 13: Proficiency Testing • Requirements All DNA technical personnel - Two external tests per year
− Approved Provider (ISO Guide 43)
Testing in each discipline on an annual basis
− Per year, at least one test for each discipline − All loci used by the laboratory must be included in the proficiency tests − Presently STRs, mtDNA − How does NGS figure in?
Standard 8: NGS Validation Developmental • Performed to validate the methodology, frequently by a
commercial or academic group
• Citations / publications in peer-reviewed journals available to
support the underlying scientific basis − Method accuracy, precision* and reproducibility* − Definition and characterization of all loci − Species specificity, sensitivity*, stability and mixtures* − Population data bases − PCR studies to assess Reaction Conditions, Multiplexing, Differential/Preferential Amplification, Product Detection
8
9/9/2014
Standard 8: NGS Validation Internal • The laboratory must internally demonstrate the reliability of a DNA procedure, which has been developmentally validated. Reproducibility Precision Sensitivity Stochastic effects Contamination assessment
TWGDAM Message: Still Relevant • Method development, data exchange, validation • Establish common controls and practices • Achieve compatibility between laboratories • Goal to create the capability to share data • Discussion and Collaboration
800.201.2011 | [email protected] | www.battelle.org 27
9
Richard A. Guerrieri Research Leader – Human Identity Battelle [email protected]
Next Generation Sequencing – Quality Assurance Considerations for the Forensic DNA Laboratory International Symposium on Human Identification Phoenix, AZ
October 2, 2014
1
Outline • Forensic DNA – where we’ve been • Next-Generation Sequencing - what does it introduce • Quality Assurance – how to navigate going forward
2
TWGDAM • Technical Working Group for DNA Analysis Methodologies
• Formed by the FBI in 1988 (now SWGDAM) • Twelve laboratories across North America
1
9/9/2014
Early Mission of TWGDAM • Early Discussion Topics RFLP
PCR
− Enzyme
DQA1, Polymarker
− Gel Lengths
D1S80 (Horizontal, Vertical Gels)
− Ethidium Bromide
STRs (Silver Stain, Fluorescence)
• How to Approach Validation – TWGDAM Guidelines − Reproducibility & Concordance − Sensitivity & Degradation − Non-Probative Samples / Mixtures
Early Mission of TWGDAM • Discussion and Collaboration Identified an objective to achieve compatibility between
laboratories
Recognized it would be necessary to establish common
controls and practices
The goal became the capability to share data
− National DNA Index System (NDIS)
DNA Identification Act of 1994 • A statute passed by Congress related to DNA testing: Authorized the formation of the DNA Advisory Board
− Goal to evaluate current technology and quality practices for forensic DNA and database laboratories − Responsibility to recommend quality assurance practices to the FBI Director for issuance as standards for DNA testing laboratories
• DNA Quality Assurance Standards formally issued to the forensic community − Forensic DNA Testing Laboratories: October 1, 1998 − Convicted Offender DNA Testing Laboratories: April 1, 1999
2
9/9/2014
DNA Quality Assurance Standards • Covers a wide range of quality affecting practices − Personnel, Facilities, Evidence Handling, Validation, Analytical Procedures, Equipment Maintenance/Calibration, Report Writing, Data Review, Audits, Proficiency Testing, Corrective Action
• Who is required to comply with the DNA QAS? − The FBI Laboratory − All laboratories that receive federal funds pursuant to the Act − Laboratories that participate in the NDIS
DNA QAS and Next-Generation Sequencing • Does next-generation sequencing (NGS) introduce any new requirements?
• Would all existing requirements directly apply? • Are the current DNA QAS relevant to NGS forensic and database applications?
8
CE
DNA Quant
Normalize
PrePCR
PCR Amp
DNA Quant
Normalize
PrePCR
PCR Amp
Inject (CE)
Interpret
NGS
Library Construction
Sequence
Interpret
9
3
9/9/2014
CE
DNA Quant
NGS
Normalize
PrePCR
PCR Amp
QA - Most Are Compatible
DNA Quant
Normalize
PrePCR
PCR Amp
Inject (CE)
Interpret
QA – Other Steps Are New
Library Construction
Sequence
Interpret
10
NGS: Key Areas to Consider • Library Construction − Controls / Quality Measures − Instrumentation / Performance Acceptance − Facilities / Work Flow
• Sequencing − Controls / Quality Measures − Reagents − Pooling / Multiplexing
• Interpretation − Thresholds − Bioinformatics − Data Basing / Expert Systems 11
How Do We Address The DNA QAS For NGS? • Standard 2 Definitions: Contamination – Unintentional introduction of exogenous DNA into
a DNA sample or PCR − Do these terms capture Library Materials?
Critical Reagents – Empirical studies required for calibration or
performance checks required prior to use − How do you conduct performance checks on sequencing reagents/instrumentation?
12
4
9/9/2014
Standard 2: Definitions • Methodology – Analytical processes/procedures used to support a DNA typing technology Extraction methods, quantitation methods
− Library Construction Methods
• Technology – The type of DNA analysis performed STR technology; Mitochondrial Sequencing Technology
− May Need NGS Term(s): Example - SNP Typing
13
Standard 2: Definitions • Positive Amplification Control – used to determine if the PCR performed properly (introduced at amplification) − Are multiple controls required? - Do you need a step to trouble-shoot the library preparation?
• Negative Amplification Control – used to detect DNA contamination of the amplification reagents − What is comparable for NGS – is a negative control required for each step (library preparation, PCR amplification, sequencing)? - What do you learn from the control at each step?
• Reagent Blank Control – used to monitor for contamination from extraction to final analysis − Is this still applicable?
14
New Terms to Consider • De-Multiplex • Index Tags / Indexing • Library • Sequencer • Others 15
5
9/9/2014
Standard 6: Facilities • Is the laboratory designed to minimize contamination? Laboratories must demonstrate a separation in time or space
for examinations, extractions and PCR setups - Amplified DNA must be generated (thermal cyclers), processed and maintained in room(s) separate from the examination, extraction and PCR setup areas.
For robotics, accommodations for analysis through
amplification in a single room, if supported by internal laboratory validation - If contamination occurs, it is minimized, addressed and less than or equal to that observed when procedure is performed manually in separate rooms.
Standard 6: Facilities • Completely separate work areas? Pre-PCR Work Post PCR and Pre-Library Work Post Library Amplification and Sequencing
• Combination of separate work areas with dedicated one-way work flow?
Standard 9: Analytical Procedures The laboratory must identify all critical reagents and detail the
QC measures in place − Pass/Fail criteria (range of acceptable results) − Procedures for acting upon unacceptable data − Documentation of the approval/rejection of data
Critical reagents and critical instrumentation require
acceptance testing prior to use on forensic/database samples
6
9/9/2014
Standard 9: Analytical Procedures So what might be an NGS critical reagent or critical instrument?
− PCR Reaction/Enzyme Mixes − Primer Sets − Purification & Normalization Beads − Thermal Cycler − Sequencing Kits
How do we define acceptance testing for NGS prior to actual
use on forensic/database samples?
Standard 9: Analytical Procedures • Does the laboratory have and follow written general guidelines for the interpretation of data? Laboratories must monitor analytical procedures using
appropriate controls and standards
This includes requiring an assessment of controls for defined
results (blanks, negative controls); correct allele types for positive controls − Assessment must be documented
Standard 9: Analytical Procedures • What might be appropriate controls and standards for NGS to monitor the analytical process?
• What would constitute defined acceptable results? Extraction reagent blanks
− Not applicable PCR no template controls
− Introduced during library preparation Positive Controls
− Introduced during library preparation
21
7
9/9/2014
Standard 9: Analytical Procedures • Other areas of the analytical method that may be important to consider monitoring? Multiplexing
− Index Tags
Interpretation
− Thresholds - Analytical and/or Stochastic - One base line for delineating noise and defining alleles? - Acceptance of increased data (+/- stutter); sequence variants
22
Standard 13: Proficiency Testing • Requirements All DNA technical personnel - Two external tests per year
− Approved Provider (ISO Guide 43)
Testing in each discipline on an annual basis
− Per year, at least one test for each discipline − All loci used by the laboratory must be included in the proficiency tests − Presently STRs, mtDNA − How does NGS figure in?
Standard 8: NGS Validation Developmental • Performed to validate the methodology, frequently by a
commercial or academic group
• Citations / publications in peer-reviewed journals available to
support the underlying scientific basis − Method accuracy, precision* and reproducibility* − Definition and characterization of all loci − Species specificity, sensitivity*, stability and mixtures* − Population data bases − PCR studies to assess Reaction Conditions, Multiplexing, Differential/Preferential Amplification, Product Detection
8
9/9/2014
Standard 8: NGS Validation Internal • The laboratory must internally demonstrate the reliability of a DNA procedure, which has been developmentally validated. Reproducibility Precision Sensitivity Stochastic effects Contamination assessment
TWGDAM Message: Still Relevant • Method development, data exchange, validation • Establish common controls and practices • Achieve compatibility between laboratories • Goal to create the capability to share data • Discussion and Collaboration
800.201.2011 | [email protected] | www.battelle.org 27
9
