Module 6: Microbial Genome Sequencing

MB 360: Scientific Inquiry in Microbiology At the Bench

Carlos C. Goller, Ph.D.

NC State University | Department of Biological Sciences

Camila Loyola, Graduate Teaching Assistant

2026-08-19

Module 6: Microbial Genome Sequencing

MB 360: Scientific Inquiry in Microbiology At the Bench

MB 360 Lab Workbook
NC State University | Department of Biological Sciences

Overview

Weeks 11–12 · DNA isolation · Quantification · Sequencing preparation · Early genome analysis

  • Connect phenotype to genotype by sequencing the isolate genome
  • Use the NO-MISS workflow for DNA extraction
  • Analyse with Oxford Nanopore sequencing, BV-BRC, and SeqHub

Learning Outcomes

  • List safety considerations for extracting DNA from microbial isolates
  • Explain the purpose of reagents used for DNA extraction
  • Discuss the significance of high-molecular-weight DNA for sequencing
  • Practice DNA extraction and Qubit fluorometer quantification
  • Collect and interpret DNA quantification and sequencing data
  • Revise the draft for the individual and group projects

Skills & Knowledge

Skills

  • Follow lab safety and PPE procedures
  • Extract and quantify microbial DNA
  • Analyse microbial DNA sequence data

Knowledge

  • PPE requirements for DNA isolation
  • DNA extraction and quantification methods
  • Oxford Nanopore Technologies sequencing workflows

Background: Sequencing Pipeline

Workflow diagram showing nanopore sequencing followed by basecalling, assembly, and genome exploration.

Nanopore sequencing and downstream analysis workflow.

NO-MISS extraction → Nanopore sequencing → BV-BRC assembly → SeqHub annotation

Lab Safety

  • Wear lab coat, goggles, and gloves
  • Clean benches and pipettors with 70% ethanol
  • Treat all culture-associated materials as biohazards
  • Decontaminate liquids, plates, and work areas at the end of the session

Methods: DNA Extraction

  1. Pellet 1.0 mL of culture in a DNA LoBind tube
  2. Resuspend in TE buffer with lysozyme
  3. Add Proteinase K and RNase A
  4. Add CTAB buffer; incubate at 56 °C for 30 minutes
  5. Combine lysate with Monarch gDNA Binding Buffer (2:1)
  6. Transfer lysate to a Monarch gDNA Column; centrifuge to bind DNA
  7. Wash with Wash Buffer (×2)
  8. Elute with 50 µL warm elution buffer (pre-heated to 65 °C)

Methods: DNA Quantification (Qubit)

  1. Prepare the Qubit working solution: 199 µL buffer + 1 µL reagent per sample
  2. Add 198 µL working solution to two standard tubes; add 2 µL of each standard
  3. Add 197 µL working solution to the sample tube; add 3 µL of your eluate
  4. Vortex all tubes; incubate 2 min in the dark
  5. Read on the Qubit fluorometer using the dsDNA BR or HS assay
  6. Record ng/µL concentration and calculate total yield

Results & Discussion

  • Record Qubit concentration and total DNA yield
  • Determine whether yield meets the sequencing threshold (≥ 400 ng total)
  • Note any low-yield samples and discuss likely causes (over-dilution, lysis inefficiency)

Reflection Questions

  1. Why is high-molecular-weight DNA important for long-read nanopore sequencing?
  2. What role does the CTAB buffer play in the NO-MISS extraction workflow?
  3. How would you troubleshoot a Qubit reading of 0 ng/µL?