The Scene

You have a task: to carefully process the samples from the scene where the individual died. You review the documentation and evidence bag. Your team is ready to learn more about the metadata (“data about data”) and what led to the passing of our beloved Squeaky Wiggles S. Honeycomb. The scene is littered with pieces of plastic, honey, and evidence of an ongoing feast.

You are able to review the security camera footage and identify Squeaky in the middle of the screen (lower left Petri dish). Squeaky is active and interacting with others but stops moving, turns dark, and eventually passes. What could have happened?

Video: Time-lapse of Squeaky and friends in their Petri dish homes. Squeaky is in the lower left Petri dish in the top right corner.

Neighbors and family members remember Squeaky fondly and do not recall any enemies. Mrs. Rollee said:

Squeaky was always willing to share their food and help with routine tasks. They were very active, ate and rested, and didn’t seem to have any health concerns. I’m shocked!

The Samples

The team has frozen samples, including tissues and feces. The samples have been meticulously collected and frozen at -80°C or immediately processed to obtain total DNA. The samples are labeled, and all metadata are carefully entered into a spreadsheet. You have access to information about the samples, including the date of collection, type of sample, and concentration (if DNA).

The Goal of the Investigation

Your goal is to use the frozen samples and total DNA to learn about the microbes and genes that could have caused the passing of Squeaky. To achieve this goal, you will work with your team to catalog, quantify, and prepare the DNA for sequencing using Oxford Nanopore Technologies (ONT). Sequencing using ONT will allow your team to obtain sequencing data quickly in your laboratory.

The PORES workshop will provide knowledge, skills, and experience that will help you gain confidence and encourage you to use ONT and long-read sequencing applications. In an effort to emphasize transparency, we will use the Transparency in Learning and Teaching (TILT) framework to include the purpose, knowledge, skills, task, and criteria for success for each activity.

Purpose

The purpose of the workshop is twofold: first, to simulate the process biomedical researchers go through in the laboratory when preparing and sequencing samples with ONT long-read sequencing, and second, to help you think about downstream applications and considerations for using ONT.

Skills

The PORES workshop will help you practice the following skills:

• Observing and describing the process of DNA extraction, quantification, assessment, and preparation of sequencing libraries.
• Formulating a hypothesis to explain a biological phenomenon
• Critical thinking and experimental interpretation
• Finding, citing, and summarizing research from a peer-reviewed scientific journal

Knowledge

This workshop will help you gain the following knowledge:

• Familiarity with the appearance and physiology of Galleria mellonella
• Understanding of the key steps for DNA extraction and preparation for sequencing with Nanopore instruments.
• Understanding of the links between microbiome and Galleria metabolism.

Task

This eBook will help you prepare for our in-person sessions, during which you will work in teams to prepare and sequence samples. You will then analyze the data and use published reliable sources to inform your next steps as you develop a hypothesis for Squeaky’s death.

Criteria for Success

While experiments do not always work, success in PORES will be measured by your ability to communicate with peers and document results in a way that emphasizes accessibility, reproducibility, and openness. For this, your team will actively contribute to this book by creating resources and sharing data. We will discuss data management and attribution to ensure we share our work responsibly and attribute sources correctly. As researchers and scholars, we strive for accuracy and specificity. Document your work carefully, and you will achieve the goals of this workshop.

Let’s begin the investigation!