Purpose

The purpose of these lab activities is to prepare DNA extracted from tissues and feces from Squeaky Wiggles S. Honeycomb. The previously quantified genomic and metagenomic DNA will be used to prepare sequencing libraries for use with Oxford Nanopore Technologies’ long-read sequencing devices.

Skills

This lab session will help you practice the following skills:

• Reading and carefully following a protocol with several different solutions and combinations.
• Critical thinking and experimental interpretation, focusing on experiments using automation.
• Documenting the procedure followed and results obtained.

Knowledge

This lab session will help you gain the following knowledge:

• Familiarity with the general steps for DNA sequencing library preparation using automation (the Miroculus Miro Canvas and the ONT VolTRAX2).
• Understanding of the functional roles of the reagents and conditions used for DNA library preparation using the ONT Ligation-based sequencing kits.
• Understanding of the advantages and disadvantages of DNA sequencing library preparation with a ligation-based approach.

Tasks

Read the protocols for the Miroculus Miro Canvas ONT library preparation and the VolTRAX2 Sequencing Kit.

We will spend time discussing the steps before loading the instruments.

Teams will work on individual samples, and the group will share and discuss the results.

Your goal is to use the DNA to learn about the microbes and wax worm genes that could have caused the passing of Squeaky. To achieve this goal, you will work with your team to prepare the DNA for sequencing using Oxford Nanopore Technologies (ONT).

Procedure

1. Read the Miroculus Miro ONT protocol.
2. Thaw the Ligation Sequencing Kit v14 and NEB Oxford Nanopore Companion kit components at room temperature.
3. Carefully label 12 PCR tubes 1-12.
4. Label two microfuge tubes, “Ethanol” and “Water.”
5. Thaw and mix the reagents as indicated by the instructors and ONT protocol.
6. Use the Miroculus Miro ONT protocol table to pipette the reagent combinations into each of the tubes. Pay close attention to the pipettes you use and ensure accurate volumes are dispensed. Note that the Drop Gloss is viscous.
7. Set up the Miro Canvas to run the ONT protocol.
8. Load the cartridge in the device.
9. Pipette each sample, one at a time, ensuring no air is injected into the cartridge. After dispensing, keep the pipette tip in the port and plunger pressed and count 1, 2, 3 before removing the tip with the pipette plunger pressed to ensure all liquid and no air enters the port.
10. After loading the Miro cartridge, start the protocol. The library prep takes about 3 hours. We will quantify the samples after the lunch break.
11. Thaw the VolTRAX Sequencing Kit reagents on ice and spin down briefly.
12. Use the VolTRAX2 software to load the reagents with p20 pipette tips as indicated.
13. Carefully insert the tip at an angle until the tip clicks into the port. Then, carefully inject the liquid, monitoring how much enters the well as indicated on the screen.
14. Start the protocol after loading all necessary reagents.
15. Clean your bench and pipettors with ethanol and tidy up your workplace.

Criteria for Success

We are using real worm samples! We will describe how they were collected and preserved for you to take notes. The amount of DNA obtained will vary depending on the sample type, the efficiency of cell lysis, and the removal of contaminants, for example. Thus, your concentrations may vary. Quality may also vary. Do not fear: you will be able to sequence samples, as we have backups and are working in teams to ensure everyone has an opportunity to prepare DNA for sequencing! The VolTRAX and Miro instruments are still in development. There are bugs and glitches. We will all try the same DNA samples to obtain libraries to sequence and have the opportunity to practice again later on. Thus, success will be running the library prep instruments and gaining experience in automated library prep.

Check Your Knowledge and Practice!