TITLE: Birchler De Allende - Using The Oxford Nanopore Ligation Sequencing Kit to Sequence Zebrafish Larvae
AUTHORS: Ian Birchler De Allende

[DESCRIPTION]
Using The Oxford Nanopore Ligation Sequencing Kit to Sequence Zebrafish Larvae


[STEPS]
SECTION: Introduction
1. 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.

SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
3. Materials For Qiagen DNeasy Blood and Tissue Kit:
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
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
4. Transfer the sample (e.g., animal tissue, cells, or larvae) to a microcentrifuge tube.
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
5. Add 180 μL Buffer ATL and 20 μL proteinase K to the sample.
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
6. 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
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
7. After lysis, vortex the sample and add 200 μL Buffer AL. Mix thoroughly.
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
8. Incubate at 70°C for 10 minutes to enhance DNA yield.
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
9. Add 200 μL ethanol (96-100%) and mix well.
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
10. Transfer the entire mixture to a DNeasy Mini spin column placed in a collection tube.
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
11. Centrifuge at 8000 rpm (6000 x g) for 1 minute. Discard the flow-through
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
12. Add 500 μL Buffer AW1 to the spin column and centrifuge for 1 minute at 8000 rpm
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
13. Add 500 μL Buffer AW2 and centrifuge for 3 minutes at 13,000 rpm (20,000 x g) to dry the membrane
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
14. Place the spin column in a clean 1.5 mL microcentrifuge tube
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
15. Add 200 μL Buffer AE directly onto the membrane.
SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
16. Incubate at room temperature for 1 minute, then centrifuge for 1 minute at 8000 rpm to elute the DNA
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
17. Start with 1 μg of high molecular weight genomic DNA.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
18. Adjust the volume to 47 μL using nuclease-free water.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
19. 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.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
20. Incubate using a thermal cycler:
20°C for 5 minutes
65°C for 5 minutes
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
21. Add 60 μL of AMPure XP beads to the reaction.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
22. Incubate at room temperature for 5 minutes.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
23. Place on a magnetic rack for 5 minutes and remove the supernatant.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
24. Wash twice with 200 μL of 70% ethanol without disturbing the beads.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
25. Air-dry the beads for 5 minutes.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
26. Elute the DNA in 60 μL of nuclease-free water.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
27. Prepare the Adapter Ligation Mix:
60 μL end-prepped DNA
25 μL Blunt/TA Ligase Master Mix
10 μL Adapter Mix (AMX)

SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
28. Mix gently by pipetting.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
29. Incubate at room temperature for 10 minutes.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
30. Add 40 μL of AMPure XP beads to the ligation reaction.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
31. Incubate and wash as in the previous clean-up step.
SECTION: Library Preparation Using the Oxford Nanopore Ligation Sequencing Kit
32. Elute in 15 μL of Elution Buffer.
SECTION: Priming and Loading the Flow Cell
33. 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.

SECTION: Priming and Loading the Flow Cell
34. 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
SECTION: 1. Quality Control using NanoPlot
38. Install NanoPlot:
 

Run NanoPlot on your FASTQ files:
 

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
SECTION: Priming and Loading the Flow Cell
35. 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.

SECTION: 3. Genome Assembly using Flye
40. Install Flye:
 

Run Flye assembly:
 

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
SECTION: Bioinformatics:
36. 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.
SECTION: Bioinformatics:
37. 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.
SECTION: 2. Optional read filtering and trimming to remove low-quality sequences or adapter contamination.
39. Adapter Trimming:
Use Porechop to remove adapter sequences from both ends of reads.
 

Use NanoFilt to filter reads based on quality and length. 

 


SECTION: 4. Variant Calling using Medaka:
41. Install Medaka:
 

SECTION: 4. Variant Calling using Medaka:
42. Run Medaka for variant calling:
 

SECTION: Extraction of DNA from Zebrafish Larvae - The QIAGEN DNeasy Blood &amp; Tissue Kit
2.