Course Information
| Course Code | Course Name | Credit Type |
|---|---|---|
| CUBI2546 | Gene Sequencing Using Nanopore Sequencer and Other Methods | (0 + 2 + 2) |
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| Course Code | Course Name | Credit Type |
|---|---|---|
| CUBI2546 | Gene Sequencing Using Nanopore Sequencer and Other Methods | (0 + 2 + 2) |
This course provides practical training in next-generation sequencing (NGS) using the Oxford Nanopore Sequencer, focusing on long-read chemistry and data analysis. Students will gain hands-on experience in library preparation, sequencing, and bioinformatics analysis.
Scale: 3 = Strong, 2 = Moderate, 1 = Slight, “–” = No Contribution
| CO / PO | PO1 | PO2 | PO3 | PO4 | PO5 | PO6 | PO7 | PO8 | PO9 | PO10 | PO11 | PO12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CO1 | 3 | 3 | 2 | 2 | 2 | 2 | 2 | – | – | – | – | – |
| CO2 | 3 | 3 | 2 | 2 | 2 | 2 | 2 | – | – | – | – | – |
| CO3 | 3 | 3 | 2 | 2 | 2 | 2 | 2 | 3 | 2 | 2 | – | – |
| CO4 | 3 | 3 | 2 | 2 | 2 | 2 | 2 | 3 | 2 | 2 | – | – |
| CO5 | 3 | 3 | 2 | 2 | 2 | 2 | 2 | 3 | 2 | 2 | – | – |
| Session No. | Module | Topic | Activities |
|---|---|---|---|
| 1 | Module I | Introduction to NGS | Overview of sequencing workflows |
| 2 | Module I | DNA Isolation – Basics | Sample collection & lysis |
| 3 | Module I | DNA Isolation – Hands-on | Precipitation, washing, elution |
| 4 | Module I | RNA Isolation | TRIzol-based extraction |
| 5 | Module I | Plasmid Extraction | Alkaline lysis protocol |
| 6 | Module I | cDNA Synthesis – Theory | Introduction to reverse transcription |
| 7 | Module I | cDNA Synthesis – Hands-on | RT-PCR workflow |
| 8 | Module I | Quantification – Qubit | DNA/RNA quantification |
| 9 | Module I | Quality Check – Gel Electrophoresis | Integrity and purity check |
| 10 | Module I | Library Prep – Overview | Steps in library construction |
| 11 | Module I | Library Prep – End Repair | End-polishing reactions |
| 12 | Module I | Adapter Ligation | ONT adapter attachment |
| 13 | Module I | Purification – Beads | AMPure XP bead cleaning |
| 14 | Module I | Library QC | Qubit & gel validation |
| 15 | Module I | Falsification Activity 1 | Compare high- vs low-quality DNA |
| 16 | Module I | NGS Troubleshooting | Sample prep errors |
| 17 | Module I | Contamination Check | Controls & blank samples |
| 18 | Module I | Best Practices | Pipetting, storage |
| 19 | Module I | Revision Session I | Recap of Module I |
| 20 | Module I | Assessment I | Short evaluation |
| 21 | Module II | MinIon Hardware Overview | Ports, components |
| 22 | Module II | Software Setup | MinKnow installation |
| 23 | Module II | Flow Cell Basics | Pore count, QC |
| 24 | Module II | Flow Cell Handling | Storage & temperature |
| 25 | Module II | Priming the Flow Cell | ONT protocols |
| 26 | Module II | Degassing | Bubble removal |
| 27 | Module II | Loading DNA | Sample drop protocol |
| 28 | Module II | Sequencing Start | Run initialization |
| 29 | Module II | Monitoring Run | Live read check |
| 30 | Module II | Stopping Sequencing | Run closure |
| 31 | Module II | Flow Cell Unloading | Washing protocol |
| 32 | Module II | Falsification Activity 2 | Flow cell handling comparison |
| 33 | Module II | Instrument Troubleshooting | Common errors |
| 34 | Module II | Best Practices | ONT guidelines |
| 35 | Module II | Run Optimization | Yield maximization |
| 36 | Module II | Re-running a Sample | Reload strategies |
| 37 | Module II | MinKnow Logs | Run files review |
| 38 | Module II | Safety & Storage | Instrument care |
| 39 | Module II | Revision Session II | Recap |
| 40 | Module II | Assessment II | Short test |
| 41 | Module III | Raw Data Handling | FAST5 basics |
| 42 | Module III | FASTQ Generation | Basecalling |
| 43 | Module III | EPI2ME Introduction | Cloud workflows |
| 44 | Module III | Genome Assembly – Basics | Long-read assembly |
| 45 | Module III | Genome Assembly – Hands-on | Flye, Miniasm |
| 46 | Module III | Transcriptome Analysis | Mapping reads |
| 47 | Module III | Differential Expression | Count matrices |
| 48 | Module III | GO Analysis | Ontologies |
| 49 | Module III | Pathway Mapping | KEGG tools |
| 50 | Module III | Protein Annotation | InterProScan |
| 51 | Module III | Sequence Alignments | BLAST, minimap2 |
| 52 | Module III | Quality Filtering | Q-score filtering |
| 53 | Module III | Visualization Tools | IGV, plots |
| 54 | Module III | Falsification Activity 3 | Compare bioinformatic outputs |
| 55 | Module III | Pipeline Automation | Snakemake basics |
| 56 | Module III | Metagenomics Intro | Species identification |
| 57 | Module III | Metagenomics – Hands-on | Kraken2, Bracken |
| 58 | Module III | Report Preparation | NGS summary reporting |
| 59 | Module III | Troubleshooting – Analysis | Common pipeline errors |
| 60 | Module III | Revision Session III | Recap of Module III |
| 61 | Module III | Practice Exam | Hands-on test |
| 62 | Module III | Doubt Clearing | Q&A session |
| 63 | Module III | Final Practical Run | Full NGS workflow simulation |
| 64 | Module III | Final Assessment | Evaluation of skills |
| Session No. | Project | Topic / Task | Outcome |
|---|---|---|---|
| 1 | Project 1 | Introduction & planning for metagenomic sequencing project | Define objectives & workflow |
| 2 | Project 1 | Sample collection & metadata recording | Understand sampling design |
| 3 | Project 1 | DNA extraction and library preparation | Prepare sequencing-ready samples |
| 4 | Project 1 | Nanopore sequencing run | Generate long-read metagenomic data |
| 5 | Project 1 | Microbial composition analysis & falsification exercise | Compare community profiles across samples |
| 6 | Project 2 | Microbe selection & project planning | Select organism & objectives |
| 7 | Project 2 | DNA extraction & QC | High-quality DNA for sequencing |
| 8 | Project 2 | Library preparation for microbial genome | NanoPore-ready library |
| 9 | Project 2 | Genome sequencing using MinIon | Obtain long-read genome data |
| 10 | Project 2 | Genome assembly, annotation & falsification exercise | Compare genomes of different strains |
| 11 | Project 3 | Experimental setup & condition selection | Define RNA-Seq experiment |
| 12 | Project 3 | RNA extraction & cDNA preparation | Generate cDNA libraries |
| 13 | Project 3 | Sequence RNA samples using MinIon | Obtain long-read transcriptomic data |
| 14 | Project 3 | Gene expression analysis & visualization | Identify DEGs & visualize results |
| 15 | Project 3 | Falsification exercise: Compare conditions | Assess expression consistency |
| 16 | Project 4 | NGS dataset retrieval from public repositories | Perform dataset mining |
| 17 | Project 4 | Identification of genes & transcription factors | Extract key regulatory elements |
| 18 | Project 4 | Functional annotation of identified genes | Annotate for biological roles |
| 19 | Project 4 | Falsification exercise: Compare datasets | Evaluate consistency across datasets |
| 20 | Project 4 | Final project presentation & report submission | Prepare & defend project outcomes |
| S.No. | Title | Author(s) / Publisher |
|---|---|---|
| 1 | Sequencing technologies – the next generation | Metzker, M.L. (2010), Nat Rev Genet 11(1):31–46 |
| 2 | Comparison of methods for differential expression analysis of RNA-seq data | Soneson, C., Delorenzi, M. (2013), BMC Bioinformatics 14:91 |
| 3 | Next Generation Sequencing and Sequence Assembly | Ali Masoudi-Nejad et al., Springer (2013) |
| 4 | Genome-wide identification & characterization of CRK gene family in cucumber | Nanda, S., Rout, P., Ullah, I. et al. (2023), BMC Genomics |
| 5 | Educational guide for nanopore sequencing in the classroom | Salazar A.N. et al. (2020), PLoS Comput Biol 16(1): e1007314 |