Clinical Applications of Nucleic Acid Amplification

Preface
Contents
Contributors
Part I: Foundations for Successful Assays
Chapter 1: A Brief Practical Guide to PCR
1 Background
2 PCR Reaction Components
2.1 DNA
2.2 DNA Polymerases
2.3 Buffer
2.4 Oligonucleotides
2.5 dNTP
3 Example Workflow for PCR
4 Perform PCR
4.1 Taq Polymerase Example
4.2 Q5 Polymerase Example
References
Chapter 2: Managing a PCR Contamination Event in a Molecular Pathology Laboratory
1 Introduction
2 Identification of a Contamination Event
3 Risk Assessment of the Contamination Event
4 Root Cause Analysis
5 Systematic Approach to Decontamination
6 Returning to Normal After a Contamination Event
References
Chapter 3: Next-Generation Sequencing Informatic Architecture Considerations
1 Introduction
2 IT Infrastructure
3 NGS Data Storage and Security
4 Disaster Recovery Plan
5 Bioinformatics Pipeline
6 Bioinformatics Pipeline Versioning
7 Conclusions
Supplemental Resources
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Part II: Applications in Oncology
Chapter 4: Nuclease Enrichment and qPCR Detection of Rare Nucleotide Variants
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Genomic DNA Preparation
2.3 NaME-PrO Enrichment
2.4 SYBR Green Real-Time Quantitative PCR
3 Methods
3.1 Cell Culture
3.2 DNA Extraction and Quantification
3.3 NaME-PrO Enrichment
3.4 SYBR Green qPCR
3.5 Results Analysis
4 Notes
References
Chapter 5: MYD88 L265P Mutation Detection by ddPCR: Recommendations for Screening and Minimal Residual Disease Monitoring
1 Introduction
2 Materials
2.1 Laboratory Setup
2.2 Instruments and Software
2.3 Reagents and Kits
3 Methods
3.1 Sample Processing
3.1.1 Plasma Selection from BCT or EDTA Tubes
3.1.2 B-Cell Collection from BM or PB EDTA
3.1.3 CSF Storage
3.1.4 gDNA Extraction (Maxwell RSC Blood DNA: AS1400)
3.1.5 cfDNA Extraction (Maxwell RSC LV ccfDNA Kit-Custom: AX1115)
3.2 ddPCR for MYD88L265P Detection
3.2.1 ddPCR Reaction Setup
3.2.2 ddPCR Analysis
3.3 Final Remarks
4 Notes
References
Chapter 6: Capture Methylation-Sensitive Restriction Enzyme Sequencing (Capture MRE-Seq) for Methylation Analysis of Highly De...
1 Introduction
2 Materials
2.1 Genomic DNA Extraction
2.2 DNA Fragmentation
2.3 Size Selection of Fragmented DNA
2.4 Library Preparation
2.5 Library QC
2.6 MRE-Seq Data Processing
3 Methods
3.1 Genomic DNA Extraction
3.2 DNA Fragmentation
3.3 Size Selection of Fragmented DNA
3.4 Library Preparation
3.5 Library Size Selection
3.6 Library QC
3.7 Next-Generation Sequencing
3.8 MRE-Seq Data Processing
4 Notes
References
Chapter 7: LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instabil...
1 Introduction
2 Materials
2.1 DNA Extraction and Quantification
2.2 DNA Standards Identification and Preparation
2.3 Assay Design
2.4 PCR Amplification
2.5 LT-RPA Amplification
2.6 Agarose Gel Electrophoresis
2.7 Sample Preparation for Capillary Electrophoresis
2.8 Sample Preparation for Next-Generation Sequencing
2.9 Microsatellite and Microsatellite Instability Genotyping
3 Methods
3.1 DNA Extraction and Quantification from Cell Lines, Blood, and Fresh Frozen and FFPE Samples
3.2 LT-RPA Assay Design and/or Selection
3.3 Selection of Microsatellites and DNA Standards
3.4 Design of LT-RPA Amplification Primers
3.5 Determination of the Optimal PCR and LT-RPA Conditions
3.6 Validation and Optimization of PCR Primers
3.7 Validation and Optimization of LT-RPA Primers
3.7.1 TwistAmp Basic Kit
3.7.2 TwistAmp Liquid Basic Kit
3.7.3 Multiplexing LT-RPA Assays
3.8 Microsatellite and Microsatellite Instability Genotyping by Capillary Electrophoresis and NGS
3.9 Capillary Electrophoresis
3.9.1 Sample Preparation
3.9.2 Capillary Electrophoresis Reaction
3.10 Next-Generation Sequencing
3.10.1 Sample Preparation
3.10.2 NGS Reaction
4 Notes
References
Chapter 8: Liquid Biopsy for Advanced Cancer: An Amplicon-Based Massively Parallel Sequencing Panel Approach to Precision Onco...
1 Introduction
2 Materials
2.1 Sample Preparation and Preservation, cfDNA Extraction, and Quantitation
2.2 Library Preparation and Sequencing: Linear PCR, Exponential PCR, Indexing PCR, Library Amplification and Purification, and...
3 Methods
3.1 Sample Preparation and Preservation
3.2 ctDNA Extraction and Quantification
3.3 Library Preparation and Sequencing
3.3.1 Linear PCR: High-Fidelity Linear Amplification (Fig. 1, step 1)
3.3.2 Purify the Linear PCR Product
3.3.3 Exponential PCR: High-Fidelity Exponential Amplification (Fig. 1, step 2; Fig. 2)
3.3.4 Purify the Exponential PCR Product
3.3.5 Indexing PCR: Library Amplification (Fig. 1, step 3)
3.3.6 Library Purification
3.3.7 Prepare and Sequence Libraries
4 Notes
References
Chapter 9: Mate Pair Sequencing: Next-Generation Sequencing for Structural Variant Detection
1 Introduction
2 Materials
2.1 Reagents
2.2 Supplies and Equipment
3 Methods
3.1 Library Preparation
3.1.1 Input DNA
3.1.2 Tagmentation Reaction Setup
3.1.3 Stop Tagmentation
3.1.4 Tagmentation Bead Cleanup
3.1.5 Strand Displacement Reaction Setup
3.1.6 Strand Displacement Bead Cleanup
3.1.7 Circularization Reaction Setup
3.1.8 Digest Linear DNA
3.1.9 Prepare Capture Beads
3.1.10 Stop Ligation
3.1.11 Shearing
3.1.12 Capture
3.1.13 End Repair
3.1.14 End Repair Bead Wash
3.1.15 A-Tail
3.1.16 Ligation
3.1.17 Ligation Bead Wash
3.1.18 Polymerase Chain Reaction (PCR)
3.1.19 Final Bead Cleanup
3.1.20 Final Quantitation
3.2 Pooling and Sequencing
3.2.1 Pooling
3.2.2 Sequencing
3.3 Bioinformatic Analysis
3.4 MPseq Visualization and Junction Analysis
4 Notes
4.1 Library Preparation Notes
4.2 Reagent Availability
4.3 Automation
4.4 Quality Control Metrics
4.5 Overall Summary
References
Part III: Applications in the Management of Organ and Stem Cell Transplantation
Chapter 10: Cell Subset-Specific Chimerism Testing by Short Tandem Repeats Analysis for Engraftment Monitoring After Hematopoi...
1 Introduction
2 Materials
2.1 Specimen Requirements
2.2 Cell Isolation and Subset Separation
2.2.1 Equipment
2.2.2 Reagents
2.2.3 Consumables
2.3 Cell Purity Assessment Using a FACS Canto II Flow Cytometer
2.3.1 Equipment
2.3.2 Reagents
2.3.3 Consumables
2.4 Genomic DNA Extraction Using QIAamp DNA Blood Mini Kit
2.4.1 Equipment
2.4.2 Reagents
2.4.3 Consumables
2.5 STR Amplification and Chimerism Testing by Capillary Electrophoresis
2.5.1 Equipment
2.5.2 Reagents
2.5.3 Consumables
3 Methods
3.1 Mononuclear Cell Isolation by Ficoll Density Gradient
3.2 Cell Subset Separation Using Miltenyi Microbeads
3.3 Preparation of Cells for FACS Analysis
3.4 FACSCanto II Startup and Plate Setup
3.5 Subset Purity Analysis
3.6 Genomic DNA Extraction
3.6.1 DNA Extraction Using QIAmp DNA Blood Mini Kit
3.6.2 DNA Specimen Dilution
3.7 STR Amplification and Chimerism Testing
3.7.1 Informative Panels for STR Amplification
3.7.2 Determining Informative STR Alleles
3.7.3 Constructing Standard Curve (for Engraftment Detection Only)
3.7.4 Setting Up for STR PCR Amplification
3.7.5 Preparing PCR Products for the Sequencer
3.7.6 Creating the Sequencing Plate Layout on the Sequencer Computer
3.7.7 Running Sequencing Plate(s)
3.7.8 Analyzing Data with GeneMapper Software v6.0
3.7.9 Calculating the Percentage of Donor Detection (for Engraftment Detection Only)
4 Notes
References
Chapter 11: Immune Cell Lineage-Specific Chimerism Testing by Next-Generation Sequencing for Engraftment Monitoring After Allo...
1 Introduction
2 Materials
2.1 Specimen Requirements
2.2 Cell Isolation and Subset Separation Using Miltenyi Microbeads
2.2.1 Equipment
2.2.2 Reagents
2.2.3 Consumables
2.3 Cell Purity Analysis Using FACSLyric Flow Cytometer
2.3.1 Equipment
2.3.2 Reagents
2.3.3 Consumables
2.4 DNA Extraction Using QIAamp DNA Blood Mini Kit
2.4.1 Equipment
2.4.2 Reagents
2.4.3 Consumables
2.5 NGS-Based Chimerism Testing
2.5.1 Equipment
2.5.2 Reagents
2.5.3 Consumables
3 Methods
3.1 Cell Separation by Ficoll Density Gradient
3.2 Cell Subset Separation Using Miltenyi Microbeads
3.3 Preparation of Cells for FACS Analysis
3.4 Cell Purity Acquisition Using the FACSLyric Flow Cytometer
3.5 Cell Purity Analysis
3.6 DNA Extraction Using QIAamp DNA Blood Mini Kit
3.7 PCR Amplification Sample Layout
3.8 PCR Amplification Setup
3.9 Post-amplification Library Magnetic Bead Purification and Cleanup
3.10 Qubit Quantification Using Qubit Fluorometer
3.11 Library Dilution and Denaturing
3.12 Loading of Library on MiSeq Sequencer
3.13 Data Export and Analysis
4 Notes
References
Part IV: Applications in Constitutional Genetics and Genomics
Chapter 12: Rapid Whole Genome Sequencing for Diagnosis of Single Locus Genetic Diseases in Critically Ill Children
1 Introduction
2 Materials
2.1 DNA Extraction and Quantification
2.2 Library Construction Preparation
2.3 Whole Genome Sequencing
2.4 Bioinformatics Pipeline (See Table 3)
2.5 Variant Interpretation
3 Methods
3.1 DNA Extraction and Quantification
3.2 Library Preparation
3.2.1 Prepare Reagents According to Table 4
3.2.2 Tagmentation of Genomic DNA (gDNA)
3.2.3 Carry out Post Tagmentation Cleanup
3.2.4 Ligation
3.2.5 Size Selection and Cleanup
3.3 Quantification of Library by qPCR
3.4 Whole Genome Sequencing
3.5 Bioinformatics Pipeline
3.6 Quality Control (QC) Metrics
3.7 Variant Interpretation
4 Notes
References
Chapter 13: EOSAL-CNV for Easy and Rapid Detection of CNVs by Fragment Analysis
1 Introduction
2 Materials
2.1 DNA Samples
2.2 PCR Reactions
2.3 Electrophoresis
3 Methods
3.1 PCR Reaction
3.2 Electrophoresis
3.3 Protocol for Working with the Capillary Sequencer
3.4 Data Extraction from GeneMapper
3.5 Analysis of the Exported Data Using EOSAL-CS
3.6 Analysis of the Exported Data with an Excel Sheet (Manually)
3.7 Identification of Small Ins/Dels
3.8 General Recommendations
4 Notes
References
Part V: Applications in Epidemiology and Public Health
Chapter 14: Rapid Cycle and Extreme Polymerase Chain Reaction
1 Introduction
2 Materials
3 Methods
3.1 Rapid Cycle PCR
3.2 Extreme PCR
4 Notes
References
Chapter 15: CRISPR-Cas12-Based Diagnostic Applications in Infectious and Zoonotic Diseases
1 Introduction
2 Materials
2.1 LAMP Amplification
2.2 Preparation of Guide RNA (sgRNA)
2.3 Preparation of Cas12b-Mediated Trans-Cleavage Reaction
3 Methods
3.1 Preparation of JEV Genomic RNA
3.2 Screening Efficient LAMP Primers
3.3 Preparation of Guide RNA (sgRNA)
3.3.1 Preparation of PCR Products for sgRNA Transcription
3.3.2 Preparation of sgRNA by In Vitro Transcription
3.3.3 Screening of Highly Efficient sgRNA
3.4 Analysis of the Sensitivity of the Two-Step HOLMESv2 Diagnosis
3.4.1 LAMP Amplification
3.4.2 Cas12b-Based Trans-Cleavage Reaction
4 Notes
References
Chapter 16: SARS-CoV-2 Sequencing for Variant Surveillance
1 Introduction
2 Materials
3 Methods
3.1 Viral RNA Extraction
3.2 cDNA Synthesis
3.3 Target Amplification
3.4 Library Preparation
3.5 Quantification
3.6 Sequencing
3.7 Data Analysis
4 Notes
References
Part VI: Applications in Resource-Limited Medicine
Chapter 17: Tuberculosis Diagnosis Using Isothermal Nucleic Acid Amplification in a Paper-and-Plastic Device
1 Introduction
2 Materials
2.1 Fabrication Materials
2.2 DNA Amplification and Detection in Tube
2.3 DNA Amplification and Detection in FLIPP-NAAT
2.4 Testing of Clinical Patient Samples in FLIPP-NAAT
3 Methods
3.1 Fabrication of FLIPP-NAAT
3.2 DNA Amplification and Detection in Tube
3.3 DNA Amplification and Detection in FLIPP-NAAT
3.4 Testing of Clinical Patient Samples in FLIPP-NAAT
4 Notes
References
Chapter 18: Loop-Mediated Isothermal Amplification on Paper Microfluidic Chips for Highly Sensitive and Specific Zika Virus De...
1 Introduction
2 Materials
2.1 Surface Decontamination Solutions and General Labware
2.2 Paper Chip Preparation
2.3 Zika and H1N1 Virus Extraction
2.4 Conventional RT-LAMP and Paper Chip RT-LAMP
2.5 Elution from Paper Chip and Gel Electrophoresis
2.6 Gel Electrophoresis
2.7 Smartphone Imaging
3 Methods
3.1 Surface Decontamination
3.2 Paper Chip Preparation
3.3 Zika and H1N1 Virus Extraction
3.4 Conventional RT-LAMP
3.5 Paper Microfluidic RT-LAMP
3.6 Elution of Amplification Product from Paper Substrate
3.7 Gel Electrophoresis
3.8 Smartphone Imaging Quantification
4 Notes
References
Chapter 19: Ultrasensitive Reverse Transcriptase Loop-Mediated Isothermal Amplification (US-LAMP)-Based Detection of Malaria I...
1 Introduction
2 Materials
2.1 Equipment
2.2 Chemicals and Consumables
2.3 Nucleic Acid Extraction Buffers
2.4 US-LAMP Reagents
3 Methods
3.1 Sample Collection
3.2 Hole Puncher Cleaning Procedure
3.3 Total Nucleic Acid Extraction from a Single DBS
3.4 US-LAMP Assay
3.5 Result Interpretation
4 Notes
References
Index