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🧬 TRACK 16 • 12 MODULAR CHAPTERS THIRD-GENERATION SEQUENCING • STRUCTURAL VARIANTS ONT R10.4.2 • PACBIO HIFI • MINIMAP2 • CUTESV • IGV
Long-Read Genomics • Structural Variant Discovery • Clinical Pharmacogenomics • Pathogen Surveillance

Long-Read (Oxford Nanopore, PacBio HiFi) Genomics & Structural Variant Discovery

End-to-End Third-Generation Workflow: Raw Signal Basecalling (Dorado/Guppy), Galaxy Minimap2 Pipelines, cuteSV/Sniffles2 Genotyping & Clinical Applications (Pharmacogenomics, Rare Diseases, Pathogens).

Master the complete third-generation sequencing analysis ecosystem. Compare Oxford Nanopore Technologies (ONT) resistive pulse sensing and PacBio SMRT circular consensus sequencing (HiFi). Process raw ionic current squiggles from POD5/FAST5 through deep neural network basecallers (Dorado SUP model with 5mC/5hmC epigenetic calling), execute reproducible Galaxy pipelines with minimap2 and samtools, inspect complex split-read breakpoints and phased haplotype blocks in IGV, discover structural variants (> 50 bp) using cuteSV and Sniffles2, and apply workflows to high-impact clinical case studies: CYP2D6 pharmacogenomics, C9orf72/HTT repeat expansions, and real-time AMR pathogen tracking.

📦 Production Long-Read & Structural Variant Stack via Conda Bioconda / Conda-Forge
# Install Third-Generation Genomics & Structural Variant Environment
conda create -n longread_env -c bioconda -c conda-forge minimap2 samtools cutesv sniffles nanoplot chopper rasusa -y
conda activate longread_env
Long-Read Genomics & Structural Variants Graphic
Curriculum Specifications Track 16
Format & Duration
Self-Paced / Guided • 12 Modular Chapters
Sequencing Chemistries
ONT R10.4.2 (Dorado SUP) • PacBio HiFi (Revio)
Computational Toolchain
Minimap2 • cuteSV • Sniffles2 • Samtools • IGV
Variant Resolution
Structural Variants (>50 bp: DEL, INS, INV, DUP, BND)
Clinical Case Targets
CYP2D6 PGx • HTT/C9orf72 Repeats • AMR Plasmids
Academic Access
100% Free & Open Access
View 12 Modules ↓
🧬 View 12 Modules Curriculum ↗

🏥 Three Benchmark Clinical Applications of Long-Read Sequencing

Phase 4 Clinical Translation
Case 1 • Pharmacogenomics (PGx)

Resolving the Complex CYP2D6 Locus

Disentangling CYP2D6 from the 94% identical CYP2D7 pseudogene; phasing whole-gene deletions (*5), tandem duplications (*1xN/*2xN), and hybrid conversions (*36-*10) for precise metabolizer status.

Case 2 • Rare Neurological Diseases

Pathogenic Tandem Repeat Expansions

Single-molecule sizing and native 5mC methylation profiling of expanded repeat loci: C9orf72 (ALS/FTD hexanucleotide), HTT (Huntington CAG), and FMR1 (Fragile X CGG).

Case 3 • Infectious Disease & Outbreaks

Real-Time Pathogen & AMR Surveillance

Rapid on-site MinION pathogen identification, full assembly of circular antimicrobial resistance (AMR) plasmids, and bacterial 6mA/4mC restriction-modification characterization.

👥

Who This Course Is For

💻
Bioinformaticians & Genomic Analysts Transitioning from short-read Illumina pipelines into third-generation single-molecule sequencing (ONT/PacBio).
🏥
Clinical Geneticists & Pathologists Resolving repeat expansions (HTT, C9orf72), pseudogene homologies (CYP2D6), and large structural rearrangements.
🦠
Microbiologists & Epidemiologists Tracking real-time hospital pathogen outbreaks, circular AMR plasmids, and native bacterial base modifications.
🎓
Genomics PhD Scholars & Postdocs Mastering neural basecalling, minimap2 alignment presets, structural variant discovery, and IGV haplotype curation.
🎯

Learning Outcomes

  • Execute Dorado transformer model basecalling from raw POD5/FAST5 signals with 5mC/5hmC methylation.
  • Compute comprehensive third-generation QC metrics including N50, Phred Q-scores, and pore health via NanoPlot.
  • Align long reads with minimap2 across ONT and PacBio presets, optimizing split-read mapping and gap costs.
  • Discover and genotype high-confidence structural variants (>50 bp) using cuteSV, Sniffles2, and AnnotSV.
  • Manually inspect complex structural breakpoints, phase haplotype blocks, and filter alignment artifacts in IGV.
  • Translate workflows to high-impact clinical benchmarks: CYP2D6 metabolizer status, repeat expansions, and AMR plasmids.
🔬 4-PHASE CURRICULUM ARCHITECTURE

Long-Read Genomics Roadmap & Benchmarks

Self-paced, hands-on masterclass curriculum structured into 4 foundational phases with production command-line scripts and benchmark Oxford Nanopore and PacBio HiFi datasets.

PHASE 1

Chemistries & Data Ecosystem

Modules 01–03 • Chemistries, Formats & GIAB

  • ONT R10.4.2 vs PacBio SMRT circular consensus (HiFi)
  • Anatomy of raw ionic current squiggles
  • FAST5 to high-speed columnar POD5 transition
  • Long-read SAM/BAM methylation tags (MM/ML)
  • Mining Genome in a Bottle (GIAB) & HPRC benchmarks
PHASE 2

Neural Basecalling & QC

Modules 04–06 • Dorado, NanoPlot & Preprocessing

  • Dorado deep neural network transformer architecture
  • Super-Accurate (SUP) basecalling with 5mC methylation
  • NanoPlot quality analytics & read length N50 metrics
  • Evaluating Phred quality score distributions
  • Read filtering with Chopper, Filtlong & Rasusa
PHASE 3

Alignment & IGV Phasing

Modules 07–09 • Minimap2 & Haplotype Curation

  • Minimap2 heuristic seeding, chaining & preset selection
  • Coordinate sorting, indexing & coverage with Samtools
  • Mosdepth high-speed regional depth calculations
  • Visualizing split-read alignments & soft clips in IGV
  • Haplotype block phasing and alignment artifact triage
PHASE 4

SV Calling & Clinical Translation

Modules 10–12 • cuteSV, Sniffles2 & Case Studies

  • Structural variant calling with cuteSV & Sniffles2
  • VCF filtering, genotyping & AnnotSV functional annotation
  • Pharmacogenomics case: CYP2D6 pseudogene resolution
  • Neurological case: C9orf72 & HTT tandem repeat sizing
  • Outbreak case: AMR plasmid assembly & tracking
COURSE CURRICULUM

12 Comprehensive Modular Chapters

All modules include Dorado recipes, minimap2 command lines, and clinical BAM/VCF benchmarks
MODULE 01 Phase 1: Chemistries & Ecosystem

Module 1: Long-Read Sequencing Chemistries: Oxford Nanopore (ONT) vs. PacBio SMRT

Comparative biophysics of third-generation sequencing: protein nanopore resistive ionic current sensors (ONT R10.4.2) vs. Zero-Mode Waveguide (ZMW) single-molecule real-time synthesis (PacBio HiFi).

🎯 Key Deliverables:
  • Nanopore vs SMRT comparative biophysical benchmark matrix
  • POD5 columnar conversion script & GIAB reference dataset download
ONT R10.4.2 PacBio HiFi POD5 FAST5 GIAB SRA
Explore Chapter 01 →
MODULE 02 Phase 1: Chemistries & Ecosystem

Module 2: Key Long-Read Data Formats: FAST5, POD5, FASTQ & SAM/BAM Specifications

Technical evolution of raw ionic current containers: from hierarchical HDF5 (FAST5) to high-speed columnar Apache Arrow (POD5), plus long-read BAM tags (MM/ML for methylation).

🎯 Key Deliverables:
  • Nanopore vs SMRT comparative biophysical benchmark matrix
  • POD5 columnar conversion script & GIAB reference dataset download
ONT R10.4.2 PacBio HiFi POD5 FAST5 GIAB SRA
Explore Chapter 02 →
MODULE 03 Phase 1: Chemistries & Ecosystem

Module 3: Navigating Public Long-Read Repositories: NCBI SRA & GIAB Consortium

Accessing benchmark datasets from the Genome in a Bottle (GIAB) consortium and Human Pangenome Reference Consortium (HPRC); programmatic retrieval via AWS Open Data and SRA toolkit.

🎯 Key Deliverables:
  • Nanopore vs SMRT comparative biophysical benchmark matrix
  • POD5 columnar conversion script & GIAB reference dataset download
ONT R10.4.2 PacBio HiFi POD5 FAST5 GIAB SRA
Explore Chapter 03 →
MODULE 04 Phase 2: Basecalling & QC

Module 4: Neural Basecalling Architecture: From Raw Signal to FASTQ with Dorado

Translating analog ionic current squiggles into digital sequence: Connectionist Temporal Classification (CTC), Transformer/CRF architectures, Super-Accurate (SUP) models, and 5mC methylation.

🎯 Key Deliverables:
  • Dorado neural basecalling execution with 5mC epigenetic flags
  • NanoPlot N50 quality report & Chopper size-selected FASTQ
Dorado SUP Guppy NanoPlot Chopper Rasusa
Explore Chapter 04 →
MODULE 05 Phase 2: Basecalling & QC

Module 5: Long-Read Quality Control: NanoPlot, Read Length Distributions & N50 Metrics

Evaluating third-generation sequencing metrics: logarithmic read length histograms, read length N50 calculation, Phred quality score distributions, and flowcell pore health analytics.

🎯 Key Deliverables:
  • Dorado neural basecalling execution with 5mC epigenetic flags
  • NanoPlot N50 quality report & Chopper size-selected FASTQ
Dorado SUP Guppy NanoPlot Chopper Rasusa
Explore Chapter 05 →
MODULE 06 Phase 2: Basecalling & QC

Module 6: Long-Read Filtering & Preprocessing: Chopper, Filtlong & Downsampling

Optimizing reads for downstream assembly and alignment: length thresholds, quality filtering with chopper, adapter trimming, and depth normalization via rasusa.

🎯 Key Deliverables:
  • Dorado neural basecalling execution with 5mC epigenetic flags
  • NanoPlot N50 quality report & Chopper size-selected FASTQ
Dorado SUP Guppy NanoPlot Chopper Rasusa
Explore Chapter 06 →
MODULE 07 Phase 3: Alignment & IGV

Module 7: Long-Read Alignment Mechanics: Minimap2 Algorithmic Presets & Galaxy Workflows

Minimizer sketching, seed-and-extend chaining, and splice-aware alignment with minimap2; constructing reproducible end-to-end Galaxy pipelines from FASTQ to indexed BAM.

🎯 Key Deliverables:
  • Coordinate-sorted BAM alignment generated via minimap2 Galaxy pipeline
  • IGV split-read visual inspection and phased haplotype block verification
Minimap2 Samtools Mosdepth IGV Browser
Explore Chapter 07 →
MODULE 08 Phase 3: Alignment & IGV

Module 8: Alignment Quality Assessment: Samtools Stats, Depth & Mosdepth Profiling

Post-alignment metrics: mapping quality distributions (MAPQ 60 vs 0), chimeric read detection, GC bias profiling, and high-speed coverage auditing with mosdepth.

🎯 Key Deliverables:
  • Coordinate-sorted BAM alignment generated via minimap2 Galaxy pipeline
  • IGV split-read visual inspection and phased haplotype block verification
Minimap2 Samtools Mosdepth IGV Browser
Explore Chapter 08 →
MODULE 09 Phase 3: Alignment & IGV

Module 9: IGV Curation for Long Reads: Split Reads, Haplotype Blocks & Technical Artifacts

Interactive manual review: configuring IGV consensus mode, inspecting supplementary split reads (SA tag), visualizing phased haplotype blocks, and identifying homopolymer artifacts.

🎯 Key Deliverables:
  • Coordinate-sorted BAM alignment generated via minimap2 Galaxy pipeline
  • IGV split-read visual inspection and phased haplotype block verification
Minimap2 Samtools Mosdepth IGV Browser
Explore Chapter 09 →
MODULE 10 Phase 4: SV Calling & Clinical

Module 10: Structural Variant Calling: cuteSV, Sniffles2 & PBSV Algorithmic Tuning

Discovery and genotyping of structural variants ($> 50\text{ bp}$): signatures, clustering algorithms, parameter optimization for Oxford Nanopore and PacBio HiFi, and GIAB benchmarking.

🎯 Key Deliverables:
  • High-confidence structural variant callset VCF with AnnotSV tiers
  • Clinical report solving CYP2D6 metabolizer status & repeat expansions
cuteSV Sniffles2 AnnotSV CYP2D6 PGx AMR Plasmids
Explore Chapter 10 →
MODULE 11 Phase 4: SV Calling & Clinical

Module 11: Structural VCF 4.2 Interpretation: INFO Fields, Filtering & Functional Annotation

Deciphering structural VCF specifications: SVTYPE, SVLEN, END, CIPOS, genotype likelihoods (DR/DV), high-confidence filtering, and AnnotSV gene dosage impact scoring.

🎯 Key Deliverables:
  • High-confidence structural variant callset VCF with AnnotSV tiers
  • Clinical report solving CYP2D6 metabolizer status & repeat expansions
cuteSV Sniffles2 AnnotSV CYP2D6 PGx AMR Plasmids
Explore Chapter 11 →
MODULE 12 Phase 4: SV Calling & Clinical

Module 12: Real-World Clinical Case Studies: Pharmacogenomics (CYP2D6), Rare Diseases & Pathogens

Translational applications: solving the polymorphic CYP2D6 star-allele puzzle, detecting pathogenic tandem repeat expansions in rare diseases, and real-time AMR pathogen surveillance.

🎯 Key Deliverables:
  • High-confidence structural variant callset VCF with AnnotSV tiers
  • Clinical report solving CYP2D6 metabolizer status & repeat expansions
cuteSV Sniffles2 AnnotSV CYP2D6 PGx AMR Plasmids
Explore Chapter 12 →
Peer-Reviewed Foundations

📚 Foundational Literature & Methodology Benchmarks

Primary sequencing chemistry papers, basecaller benchmarks & SV algorithms for Track 16

🧬 Long-Read Chemistries, Neural Basecalling & Alignment

  • Jain M, Koren S, Miga KH, et al. (2018). Nanopore sequencing and assembly of a human genome with ultra-long reads. Nature Biotechnology 36(4):338–345. doi:10.1038/nbt.4060
  • Wenger AM, Peluso P, Rowell WJ, et al. (2019). Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nature Biotechnology 37(10):1155–1162. doi:10.1038/s41587-019-0217-9
  • Li H. (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34(18):3094–3100. doi:10.1093/bioinformatics/bty191
  • De Coster W, D'Hert S, Schultz DT, et al. (2018). NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34(15):2666–2669. doi:10.1093/bioinformatics/bty149

🔬 Structural Variant Discovery & Clinical Translation

  • Jiang T, Liu Y, Jiang Y, et al. (2020). Long-read-based human genomic structural variation detection with cuteSV. Genome Biology 21:189. doi:10.1186/s13059-020-02107-y
  • Smolka S, Paulin LF, Grochowski CM, et al. (2024). Detection of structural variants from long-read data using Sniffles2. Nature Biotechnology 42:610–618. doi:10.1038/s41587-023-02024-y
  • Robinson JT, Thorvaldsdóttir H, Winckler W, et al. (2011). Integrative Genomics Viewer. Nature Biotechnology 29:24–26. doi:10.1038/nbt.1754
  • English AC, Salerno WJ, Hampton OA, et al. (2015). Assessing structural variation in a personal genome—towards a human genomes standard. BMC Genomics 16:286. doi:10.1186/s12864-015-1479-3

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