Oct 01, 2024
Birchler De Allende - Using The Oxford Nanopore Ligation Sequencing Kit to Sequence Zebrafish Larvae
- 1North Carolina State University
Protocol Citation: Ian Birchler De Allende 2024. Birchler De Allende - Using The Oxford Nanopore Ligation Sequencing Kit to Sequence Zebrafish Larvae. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8denv5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 01, 2024
Last Modified: October 01, 2024
Protocol Integer ID: 108735
Abstract
Using The Oxford Nanopore Ligation Sequencing Kit to Sequence Zebrafish Larvae
Introduction
Introduction
The protocols described outline methods for extracting high-quality genomic DNA from zebrafish larvae for sequencing applications. The process typically begins with collecting a small tissue sample, such as a fin clip, from 3-day post-fertilization larvae. The QIAGEN DNeasy Blood & Tissue Kit is commonly used for DNA extraction. The procedure involves tissue lysis with proteinase K, followed by binding of DNA to the silica membrane, washing steps to remove contaminants, and elution of pure DNA. This method yields PCR-ready genomic DNA suitable for various downstream applications, including genotyping and next-generation sequencing.
Following DNA extraction, library preparation for sequencing may be performed using kits like the Oxford Nanopore Ligation Sequencing Kit, which involves DNA repair, end-preparation, and adapter ligation steps. These methods enable researchers to efficiently genotype and sequence zebrafish larvae, facilitating early identification of desired genotypes for disease modeling and other genetic studies.
Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood & Tissue Kit
Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood & Tissue Kit
Figure 1. Basic steps for QIAGEN DNeasy Blood & Tissue Kit zebrafish larvae tail clip DNA extraction. Biorender Image made by Ian Birchler De Allende.
Zebrafish larvae (2-5 days post-fertilization recommended)
Proteinase K
Lysis buffer (e.g., ATL buffer for Qiagen kits)
Ethanol (96-100%)
Microcentrifuge tubes (1.5 mL)
Vortex mixer
Water bath or incubator (set to 56°C)
Nuclease-free water or TE buffer
Transfer the sample (e.g., animal tissue, cells, or larvae) to a microcentrifuge tube.
Add 180 μL Buffer ATL and 20 μL proteinase K to the sample.
Vortex thoroughly and incubate at 56°C for 1-3 hours , depending on the sample type.
For tough tissues, mechanical disruption using a bead mill or rotor-stator homogenizer can improve lysis efficiency
After lysis, vortex the sample and add 200 μL Buffer AL. Mix thoroughly.
Incubate at 70°C for 10 minutes to enhance DNA yield.
Add 200 μL ethanol (96-100%) and mix well.
Transfer the entire mixture to a DNeasy Mini spin column placed in a collection tube.
Centrifuge at 8000 rpm (6000 x g) for 1 minute. Discard the flow-through
Add 500 μL Buffer AW1 to the spin column and centrifuge for 1 minute at 8000 rpm
Add 500 μL Buffer AW2 and centrifuge for 3 minutes at 13,000 rpm (20,000 x g) to dry the membrane
Place the spin column in a clean 1.5 mL microcentrifuge tube
Add 200 μL Buffer AE directly onto the membrane.
Incubate at room temperature for 1 minute, then centrifuge for 1 minute at 8000 rpm to elute the DNA
Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
Start with 1 μg of high molecular weight genomic DNA.
Adjust the volume to 47 μL using nuclease-free water.
Prepare the End-Repair and dA-Tailing reaction:
47 μL DNA sample
3 μL NEBNext FFPE DNA Repair Buffer
2 μL NEBNext FFPE DNA Repair Mix
3.5 μL NEBNext Ultra II End Prep Reaction Buffer
Mix thoroughly by flicking the tube or gentle pipetting.
Incubate using a thermal cycler:
20°C for 5 minutes
65°C for 5 minutes
Add 60 μL of AMPure XP beads to the reaction.
Incubate at room temperature for 5 minutes.
Place on a magnetic rack for 5 minutes and remove the supernatant.
Wash twice with 200 μL of 70% ethanol without disturbing the beads.
Air-dry the beads for 5 minutes.
Elute the DNA in 60 μL of nuclease-free water.
Prepare the Adapter Ligation Mix:
60 μL end-prepped DNA
25 μL Blunt/TA Ligase Master Mix
10 μL Adapter Mix (AMX)
Mix gently by pipetting.
Incubate at room temperature for 10 minutes.
Add 40 μL of AMPure XP beads to the ligation reaction.
Incubate and wash as in the previous clean-up step.
Elute in 15 μL of Elution Buffer.
Priming and Loading the Flow Cell
Priming and Loading the Flow Cell
Priming:
Prepare the flow cell by attaching it to the MinION device.
Gently flush the flow cell with 800 µL of priming mix (provided in the sequencing kit) using the flow cell priming port.
Sample Loading:
Mix the eluted DNA with 37.5 µL of Running Buffer with Fuel Mix (RBF), and 25.5 µL of Loading Beads (LB).
Gently pipette the mixture into the flow cell via the sample loading port.
Start Sequencing:
Close the lid of the flow cell and initiate sequencing using the MinKNOW software.
Sequencing and Data Analysis
Run MinION:
Monitor sequencing through the MinKNOW software, ensuring that the flow cell performs optimally and tracks data output.
Sequencing runs can range from a few hours to days, depending on the required depth and application.
Bioinformatics:
Bioinformatics:
Key file types produced by Nanopore sequencing:
- POD5 files: These contain the raw signal data from the nanopore sequencing process. POD5 is the newer format that has replaced the legacy FAST5 format. It’s more efficient for storage and data access.
- FASTQ files: These contain the base-called sequence data, which is the raw signal converted into nucleotide sequences. FASTQ is a standard format for storing sequence data along with quality scores.
- BAM files: These are used for storing alignment information when reads are mapped to a reference genome. BAM files are also used for storing information about modified bases when performing modified base calling.
- Sequencing summary file: This contains metadata and summary statistics for the sequencing run, including information about each read such as read length, quality scores, and other metrics.
The bioinformatics workflow for nanopore data typically involves several key steps:
- Quality control using tools like NanoPlot to assess read quality, length distribution, and other metrics.
- Optional read filtering and trimming to remove low-quality sequences or adapter contamination.
- Genome assembly using long-read assemblers such as Flye for de novo projects, or alignment to a reference genome using tools like Minimap2.
- Variant calling with specialized tools like Medaka, which can also detect structural variants and methylation patterns.
1. Quality Control using NanoPlot
1. Quality Control using NanoPlot
Install NanoPlot:
pip install NanoPlot
- Run NanoPlot on your FASTQ files:
NanoPlot --fastq sample.fastq -o nanoplot_output
Examine the output in the nanoplot_output directory, including:
- NanoPlot-report.html: Interactive report with plots and statistics
- Summary statistics in .txt files
- Various plots as .png files showing read length distribution, quality scores, etc.
Key metrics to assess:
- N50 read length
- Mean read quality
- Total bases sequenced
- Read length distribution
2. Optional read filtering and trimming to remove low-quality sequences or adapter contamination.
2. Optional read filtering and trimming to remove low-quality sequences or adapter contamination.
Adapter Trimming:
Use Porechop to remove adapter sequences from both ends of reads.
porechop -i raw_reads.fastq -o trimmed_reads.fastq
Use NanoFilt to filter reads based on quality and length.
cat trimmed_reads.fastq | NanoFilt -q 10 -l 1000 > filtered_reads.fastq
3. Genome Assembly using Flye
3. Genome Assembly using Flye
Install Flye:
conda install -c bioconda flye
Run Flye assembly:
flye --nano-raw reads.fastq --out-dir flye_assembly --threads 16 --genome-size 5m
Adjust threads and estimated genome size as needed.
Examine the output:
- assembly.fasta: Final assembled contigs
- assembly_graph.gfa: Assembly graph
- assembly_info.txt: Statistics on contigs
Assess assembly quality:
- Check N50 contig length
- Examine total assembly size vs expected genome size
- Look for potential misassemblies or contamination
4. Variant Calling using Medaka:
4. Variant Calling using Medaka:
Install Medaka:
pip install medaka
Run Medaka for variant calling:
medaka_variant -i alignment.sorted.bam -f reference.fasta -o medaka_variants