Nanopore Sequencing: Methods and Protocols

Preface
Contents
Contributors
Part I: Nanopore Sequencing for Genomics and Beyond
Chapter 1: The Current State of Nanopore Sequencing
1 Introduction
2 The Technology
3 The Platform
4 Applications
4.1 The World´s Fastest Genomes
4.2 The World´s Largest Genomes
4.3 The World´s Most Complete Human Genome
4.4 Viral Genomes the World Over
4.5 Targeted Sequencing on the Other Side of the World
4.6 A World of Potential
5 Summary
References
Chapter 2: Hybrid Genome Assembly of Short and Long Reads in Galaxy
1 Introduction
2 Materials
2.1 Long- and Short-Read Datasets
2.2 Public Galaxy Instance (NanoGalaxy)
3 Methods
3.1 Basic Usage of Galaxy
3.2 Example Using Public Data
3.2.1 Analysis Overview
3.2.2 Hands-On: Obtain Sample Data from SRA
3.2.3 Hands-On: Quality Control of Nanopore Reads
3.2.4 Hands-On: Quality Filtering of Long-Read Sequencing Data
3.2.5 Hands-On: Assembly of Long-Read Sequencing Data
3.2.6 Hands-On: Quality Control of Short-Read Sequencing Data
3.2.7 Hands-On: Mapping Short Reads to Assembly for Polishing
3.2.8 Hands-On: Evaluate the Quality of Aligned Reads Data in BAM Format
3.2.9 Hands-On: Polish Assembly
3.2.10 Hands-On: Genome Assembly Metrics
4 Notes
References
Chapter 3: Microbial Genome Sequencing and Assembly Using Nanopore Sequencers
1 Introduction
2 Materials
2.1 Sequencing Reagents
2.2 Bioinformatics Tools
3 Methods
3.1 gDNA Extraction
3.2 Sequencing Library Preparation
3.2.1 DNA Repair and End Prep
3.2.2 Adapter Ligation and Cleanup
3.3 Nanopore Sequencing
3.4 Bioinformatics
4 Notes
References
Chapter 4: De Novo Genome Assembly of Japanese Black Cattle as Model of an Economically Relevant Animal
1 Introduction
2 Materials
2.1 Sample Preparation [Experiment]
2.2 Consumables and Equipment [Experiment]
2.3 Software [Data Analysis]
3 Methods
3.1 Experimental Procedures
3.1.1 Genomic DNA Extraction from Frozen Semen and DNA Quality Check
3.1.2 Long-Read Sequencing by PromethION
3.1.3 Short-Read Sequence by Illumina Sequencer
3.2 Bioinformatics Procedures
3.2.1 Outline
3.2.2 Code (See Note 13)
3.3 Analysis Example
3.4 Conclusions
4 Notes
References
Chapter 5: How to Sequence and Assemble Plant Genomes
1 Introduction
2 Materials
2.1 DNA Isolation
2.2 Size Selection
2.3 Library Prep
2.4 Draft Assembly
2.5 Assembling Organellar Genomes
2.6 Re-bridging Nuclear Genome
3 Methods
3.1 DNA Extraction
3.2 Size Selection
3.3 Library Prep
3.4 Draft Assembly
3.5 Assembling Organellar Genomes
3.6 Re-bridge the Nuclear Genome
4 Notes
References
Chapter 6: Detection of DNA Modification Using Nanopore Sequencers
1 Introduction
2 Comparison Method
3 Model-Based Method
4 Expanded Basecalling Method
5 Materials
6 Methods
7 Notes
References
Chapter 7: Ultralow-Input Genome Library Preparation for Nanopore Sequencing with Droplet MDA
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 8: The Method of Eliminating the Wolbachia Endosymbiont Genomes from Insect Samples Prior to a Long-Read Sequencing
1 Introduction
2 Materials
3 Methods
3.1 Rearing Host Spiders and the Parasitoid Larvae Until Adult Emergence (in the Case of R. nielseni, the Host Spiders Parasit...
3.2 Rearing the Adult Wasps for Sterilization of Wolbachia (in the Case of R. nielseni)
3.3 Homogenizing the Wasps, DNA Extraction, qPCR, and Evaporation
4 Notes
References
Chapter 9: A Nanopore Sequencing Course for Graduate School Curriculum
1 Introduction
2 Materials
2.1 Wet Lab Experiment Part
2.2 Bioinformatics Part
3 Methods
3.1 Genomic DNA Extraction
3.2 Nanopore Sequencing
3.3 Genome Assembly and Annotation
3.4 Genome Report
4 Notes
References
Part II: Analysis of Repetitive Regions and Structural Variants
Chapter 10: A Guide to Sequencing for Long Repetitive Regions
1 Introduction
2 Materials
2.1 Sample Homogenization
2.2 Nucleotide Extraction, Quantification, and Qualification
2.3 Sequencing Instruments and Library Prep Kits
2.4 PC Spec and Software
3 Methods
3.1 High-Molecular-Weight (HMW) Genomic DNA (gDNA) Isolation
3.2 Total RNA Extraction
3.3 gDNA Sequencing with a Nanopore Sequencer
3.4 Direct RNA Sequencing with a Nanopore Sequencer
3.5 cDNA Sequencing with an Illumina Sequencer
3.6 Bioinformatics Analysis
3.7 Filtering or Trimming of Sequence Reads
3.8 Terminal Domain Contig (as Seed Contig) Preparation
3.9 Collection of Repeat Units by Elongation
3.10 Scaffolding and Reordering of Repeat Unit Using Full-Length Long Read
3.11 Visualization of Repetitive Gene Architecture
4 Notes
References
Chapter 11: Analysis of Tandem Repeat Expansions Using Long DNA Reads
1 Introduction
2 Materials
2.1 Library Preparation for Long-Read Sequencing
2.2 Data Analysis
3 Methods
3.1 Designing gRNAs
3.2 Sequence Library Preparation
3.3 Data Analysis
3.3.1 Aligning Long Reads to the Reference Genome
3.3.2 Detect Changes in the Copy Number of Repeats with Tandem-Genotypes
3.3.3 Creating a Plot
3.3.4 Merge Reads to Create a Consensus Sequence
4 Notes
References
Chapter 12: Finding Rearrangements in Nanopore DNA Reads with LAST and dnarrange
1 Introduction
1.1 Examples of Match, Mismatch, Insertion, and Deletion Rates
1.2 Understanding Rearrangements
1.3 Simple Sequences
2 Methods
2.1 Installation
2.2 Getting the Camponotus Rates
2.3 Getting the Plasmodium falciparum Rates
2.4 Getting the Human Rates
2.5 Aligning Human DNA Reads to a Human Genome
2.6 An Alternative Way Using Windowmasker
2.7 Finding Rearrangements with dnarrange
2.8 Making Dotplot Figures of the Rearrangements
2.9 last-dotplot
2.10 Rearrangement Types and Thresholds
2.11 Other Features of dnarrange
3 Notes
References
Chapter 13: Long-Read Whole-Genome Sequencing Using a Nanopore Sequencer and Detection of Structural Variants in Cancer Genomes
1 Introduction
2 Materials
2.1 Sample Preparation (Experiment)
2.2 Consumables (Experiment)
2.3 Equipment (Experiment)
2.4 Dataset (Data Analysis)
2.5 Software (Data Analysis)
2.6 Reference Genome (Data Analysis)
3 Methods
3.1 HMW DNA Extraction (Experiment)
3.2 Library Preparation and Sequencing (Experiment)
3.3 Basecalling (Data Analysis)
3.4 Mapping of Sequencing Reads to the Reference Genome (Data Analysis)
3.5 SV Detection (Data Analysis)
3.6 Visualization and Interpretation of the Detected SVs (Data Analysis)
4 Notes
References
Part III: Rapid On-Site Microbial Detection and Epidemiology
Chapter 14: Full-Length 16S rRNA Gene Analysis Using Long-Read Nanopore Sequencing for Rapid Identification of Bacteria from C...
1 Introduction
2 Materials
2.1 General Laboratory Supplies
2.2 Sample Preparation
2.3 DNA Extraction
2.4 PCR
2.5 PCR Cleanup
2.6 DNA Quantification
2.7 Library Construction and Nanopore Sequencing
3 Methods
3.1 Preparation of Clinical Samples
3.1.1 Preparing Fecal Samples
3.1.2 Preparing Sputum Samples
3.1.3 Preparing Swab Samples
3.1.4 Preparing Whole Blood Samples
3.2 Bacterial Cell Disruption by Bead Beating
3.3 Automated DNA Purification Using Maxwell RSC System
3.4 Two-Step PCR
3.4.1 First PCR: Amplification of the 16S rRNA Gene
3.4.2 Second PCR
3.5 PCR Cleanup
3.6 DNA Quantification
3.7 Sequencing Library Preparation
3.8 Nanopore Sequencing
3.9 Bioinformatics Analysis
3.9.1 Taxonomic Classification Using EPI2ME Fastq 16S Workflow
3.9.2 Consensus Calling for Nanopore Sequencing Reads
4 Notes
References
Chapter 15: Nanopore Sequencing Data Analysis of 16S rRNA Genes Using the GenomeSync-GSTK System
1 Introduction
2 Materials
3 Methods
3.1 Preparing the Database
3.2 Preparing the Pipeline
3.3 Example of GenomeSync-GSTK Analysis
4 Notes
References
Chapter 16: Genomic Epidemiological Analysis of Antimicrobial-Resistant Bacteria with Nanopore Sequencing
1 Introduction
2 Materials and Methods
2.1 High Molecular Weight (HMW) of Bacterial Genomic DNA (gDNA)
3 Methods
3.1 Construction of Bacterial Complete Genomes with Nanopore Sequencing Data
3.2 Detection and Classification of Core Genes, Accessory Genes, and MGEs in Bacterial Genomes
3.3 Genomic Epidemiological Analysis of AMR Bacterial Isolates Using Their Genomes
4 Notes
References
Chapter 17: Rapid and Comprehensive Identification of Nontuberculous Mycobacteria
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables for Library Preparation and MinION Sequencing
2.3 Software Requirements for Computational Analysis
3 Methods
3.1 DNA Extraction from Culture
3.2 Preparing Library and Sequencing
3.3 Computational Analysis
4 Notes
References
Part IV: Nanopore Sequencing for Transcriptomics and Beyond
Chapter 18: Long-Read Single-Cell Sequencing Using scCOLOR-seq
1 Introduction
2 Materials
2.1 Oligonucleotides
2.2 Oligonucleotide Bead Design
2.3 Cell Encapsulation
2.4 Emulsion Breaking and Reverse Transcription
2.5 Exonuclease Digestion and SMART PCR
2.6 Nanopore Library Preparation and Sequencing
3 Methods
3.1 Cell Encapsulation
3.2 Emulsion Breaking
3.3 Reverse Transcription
3.4 Exonuclease Digestion
3.5 SMART PCR
3.6 Nanopore Library Preparation
3.7 Sequencing
4 Notes
References
Chapter 19: Unfolding the Bacterial Transcriptome Landscape Using Oxford Nanopore Technology Direct RNA Sequencing
1 Introduction
2 Materials
2.1 Sequencing Bacterial RNA
2.2 Software
2.3 System Requirement
3 Methods
3.1 Ribosomal RNA (rRNA) Depletion
3.2 Polyadenylation
3.3 Direct RNA-Seq
3.4 Priming and Loading the Flow Cell
3.5 UNAGI Pipeline
3.6 Running UNAGI
4 Notes
References
Chapter 20: Nanopore Direct RNA Sequencing of Monosome- and Polysome-Bound RNA
1 Introduction
2 Materials
2.1 Preparation of Whole Cell Extracts
2.2 Polysome Fractionation
2.3 RNA Purification from Sucrose Fractions
2.4 Direct RNA Sequencing
2.5 Bioinformatics Analysis
3 Methods
3.1 Preparation of Whole Cell Extracts (WCE)
3.2 Polysome Fractionation
3.3 RNA Extraction and Enrichment
3.4 Library Preparation for Direct RNA Sequencing
3.5 Bioinformatics Analysis
4 Notes
References
Chapter 21: RNA Modification Detection Using Nanopore Direct RNA Sequencing and nanoDoc2
1 Introduction
2 Materials
2.1 RNA-Sequence Data
2.2 Reference Data
2.3 Computational Environment
3 Method
3.1 Overview
3.1.1 Signal Resquiggling by Viterbi Using Trace Value
3.1.2 Training Using the Deep One-Class Algorithm
3.1.3 RNA Modification Detection Using Clustering
3.2 Data Preparation
3.3 Installation and Preparation of nanoDoc2
3.4 Inferring Modifications with nanoDoc2 Using Previously Trained Model
3.4.1 Mapping and Resquiggling
3.4.2 Modification Detection
3.4.3 Interpretation of Output
3.5 Training nanodoc2 for IVT Data
3.5.1 Prepare Training Dataset for Initial Training
3.5.2 Pre-training Model for 6-Mer Classification
3.5.3 Deep One-Class Classification for Each 6-Mer
4 Notes
References
Index