Gene Sequencing Using Nanopore Sequencer and other Methods – CUBI2546

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Dr. Polaki Suman

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Course Information

Course Code Course Name Credit Type
CUBI2546 Gene Sequencing Using Nanopore Sequencer and Other Methods (0 + 2 + 2)

Course Overview

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.

Prerequisites

  • Basic knowledge of molecular biology and genetics.
  • Familiarity with laboratory techniques in DNA/RNA handling.

Course Objectives

  • Perform next-generation sequencing (NGS) using the Oxford Nanopore Sequencer. (L3)
  • Understand the principles and applications of long-read chemistry in NGS. (L2)
  • Analyze the NGS data generated through Nanopore sequencing. (L4)

Course Outcomes

  • Understand and prepare DNA/cDNA libraries required for NGS. (L2)
  • Analyze library quality using Qubit spectrophotometry. (L3)
  • Set up the MinIon Nanopore Sequencer. (L3)
  • Manage and handle flow cells and load nucleic acid samples for sequencing. (L4)
  • Analyze NGS data and interpret results. (L5)

CO–PO Mapping

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

Practice Syllabus

🔷 Module I: Introduction to Next Generation Sequencing (NGS)

  • Introduction to Next Generation Sequencing (NGS)
  • Isolation of DNA/RNA/plasmid for sequencing
  • cDNA synthesis
  • Quantification and quality estimation of nucleic acids
  • Library preparation

Falsification Exercise:

  • Hypothesis: The quality of DNA/RNA does not affect the accuracy of NGS results.
  • Exercise: Compare NGS results from high-quality and low-quality DNA/RNA samples to evaluate the impact on sequencing accuracy.

🔷 Module II: Instrumentation of MinIon Nanopore Sequencer

  • Instrumentation of MinIon Nanopore Sequencer
  • Setting up MinIon and associated peripherals
  • Flow cell loading and unloading
  • Degassing and sample loading onto the flow cell
  • Sequencing using MinIon

Falsification Exercise:

  • Hypothesis: Flow cell handling and loading techniques do not affect sequencing performance.
  • Exercise: Test different flow cell handling and loading techniques to determine their impact on sequencing efficiency and accuracy.

🔷 Module III: Data Collection and Analysis

  • Raw data collection and format
  • NGS Workflow Overview
  • Introduction to EPI2ME
  • Genome and Transcriptome Assembly
  • Sequence Alignments and Searching
  • Pathway and GO annotation systems
  • Graphics and Data Visualization

Falsification Exercise:

  • Hypothesis: Different bioinformatics tools yield the same results for genome and transcriptome assembly.
  • Exercise: Use multiple bioinformatics tools to assemble the same dataset and compare the results for consistency and accuracy.

Projects

🔷 Project 1: Nanopore-based Genome Sequencing of Metagenomic Samples

  • Collect metagenomic samples.
  • Perform DNA extraction and library preparation.
  • Sequence the samples using MinIon.
  • Analyze sequencing data and identify microbial species.

Falsification Exercise:

  • Hypothesis: The microbial composition inferred from sequencing data is consistent across samples from the same environment.
  • Exercise: Compare sequencing results from multiple samples of the same environment to evaluate consistency.

🔷 Project 2: Genome Sequencing and Analysis of an Important Microbe

  • Select a microbe of interest (bacteria/fungi).
  • Perform DNA extraction and library preparation.
  • Sequence the genome using MinIon.
  • Assemble and annotate the genome.

Falsification Exercise:

  • Hypothesis: Different strains of the same microbial species have identical genomes.
  • Exercise: Sequence and compare genomes of multiple strains to detect variations.

🔷 Project 3: Gene Expression Analysis and Visualization from NGS Data

  • Collect RNA samples from different conditions.
  • Perform cDNA synthesis and library preparation.
  • Sequence the samples using MinIon.
  • Analyze gene expression levels and visualize results.

Falsification Exercise:

  • Hypothesis: Gene expression profiles remain identical under different experimental conditions.
  • Exercise: Compare gene expression across conditions to identify differentially expressed genes.

🔷 Project 4: Genome-wide Survey of Genes/Transcription Factors via NGS Data Mining

  • Collect NGS datasets from public databases.
  • Perform data mining to identify genes and transcription factors.
  • Analyze functional roles of identified genes.

Falsification Exercise:

  • Hypothesis: Data mining results are consistent across all NGS datasets.
  • Exercise: Analyze multiple datasets and compare results to test consistency.

Practice Session Plan – Nanopore Sequencing

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

Project Session Plan – Nanopore Sequencing

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

📚 Suggested Readings

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

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