33 DNA Lab: Hands-On Laboratory Sequencing
This module outlines the practical workflow for molecular analysis in tropical biodiversity research, tracking the process from sample collection in the field to sequence repository generation. The laboratory activities focus on DNA barcoding of the Cytochrome c Oxidase Subunit I (CO1) gene using Oxford Nanopore Technologies (ONT).
This laboratory session is broadcast live for hybrid participation. Please join using the link or credentials below:
- Meeting ID:
485 995 877 261 32 - Passcode:
3LL38TY2
33.1 1. DNA Extraction and Quality Control
Successful genomic analysis relies on extracting high-yield, high-purity, and intact genomic DNA (gDNA).
33.1.1 DNA Extraction
The extraction process isolates genetic material through three sequential phases:
- Lysis: Physical or chemical disruption of cell walls and membranes to release cellular contents. For environmental or microbiome samples, physical bead-beating is employed.
- Precipitation: Separation of DNA from proteins and other cellular debris using alcohol (ethanol or isopropanol).
- Purification: Washing and re-dissolving the DNA in DNase/RNase-free water or elution buffer for downstream applications.
The practical session utilizes the ZymoBIOMICS™ DNA Miniprep Kit to extract DNA suitable for metagenomic and microbiome analyses.
33.1.2 DNA Quality Control (QC)
DNA quality is assessed across three primary parameters:
| Parameter | Method | Target Values | Notes |
|---|---|---|---|
| Concentration | Fluorometry (Qubit) | Dependent on application | Measures concentration specifically using target-binding dyes; more accurate than spectrophotometry for low concentrations. |
| Purity (A260/A280) | Spectrophotometry (NanoDrop) | 1.8 – 2.0 | Ratios below 1.8 indicate protein or phenol contamination. Ratios above 2.0 may indicate RNA contamination. |
| Purity (A260/A230) | Spectrophotometry (NanoDrop) | 2.0 – 2.2 | Ratios below 2.0 indicate residual salts, EDTA, phenol, or carbohydrate carryover. |
| Integrity | Automated Electrophoresis (TapeStation) | DNA Integrity Number (DIN): 1 to 10 | DIN 10 represents fully intact gDNA; DIN 1 represents highly degraded DNA. |
33.2 2. Polymerase Chain Reaction (PCR) and Electrophoresis
Following extraction and quality confirmation, target marker genes are amplified and verified.
33.2.1 PCR Amplification of the CO1 Gene
The Cytochrome c Oxidase Subunit I (CO1) mitochondrial gene serves as the standard barcode marker for animal specimens. Amplification is performed using the KOD One PCR Master Mix and Folmer primers.
The thermal cycler profile is configured as follows:
| Step | Temperature | Duration | Cycle(s) |
|---|---|---|---|
| Pre-denaturation | 95°C | 3 minutes | 1 |
| Denaturation | 95°C | 30 seconds | 40 |
| Annealing | 51°C | 30 seconds | |
| Extension | 72°C | 1 minute 30 seconds | |
| Post-Extension | 72°C | 5 minutes | 1 |
33.2.2 Agarose Gel Electrophoresis
Gel electrophoresis separates DNA fragments by size to confirm successful target amplification.
- Matrix: A 1% agarose gel prepared in 1X TAE buffer.
- Migration: DNA carries a net negative charge due to its phosphate backbone and migrates toward the positive electrode (anode) when an electrical current (100V for 15 minutes) is applied.
- Visualization: Stained with a fluorescent DNA-binding dye and visualized on a blue light transilluminator. Fragment sizes are estimated by comparison against a reference DNA ladder.
33.3 3. Oxford Nanopore Technologies (ONT) Sequencing
Nanopore sequencing measures changes in electrical current as single strands of DNA pass through synthetic nanopores embedded in a membrane.
33.3.1 Library Preparation
Amplicons are prepared for sequencing using the Ligation Sequencing Amplicons – Native Barcoding Kit 96 V14 (SQK-NBD114.96). The workflow consists of three stages:
- End Prep (End-Repair and dA-Tailing): Enzymes blunt-end and phosphorylate DNA fragments, then add a single adenine (A) overhang at the 3’ end. This prepares the fragment for adapter attachment and prevents self-ligation.
- Barcode Ligation: Unique short DNA sequences (barcodes) are ligated to each sample using T4 DNA Ligase. This allows multiplexing (pooling multiple samples into a single run) to optimize flow cell usage.
- Adapter Ligation: Sequencing adapters, pre-loaded with a motor protein, are ligated to the barcoded DNA. The motor protein regulates the speed at which DNA translocates through the nanopore.
33.3.2 Flow Cell Loading and Run Initialization
The prepared library is loaded onto the MinION flow cell:
- Priming: The flow cell is primed with a priming mix (Flow Cell Flush and Flow Cell Tether) to stabilize the ionic current across the membrane.
- Loading: The prepared library is loaded slowly into the flow cell inlet port to avoid bubble formation, which can damage active pores.
- Basecalling: The MinKNOW software monitors pore activity and records ionic current changes in real time. The raw signal (“squiggle” data) is processed into basecalled sequences (FASTQ or BAM format) using the integrated Dorado basecaller.
Sequencing Module Document
The complete laboratory sequencing guidebook is available below for reading and download: