Illumina Experts: Preventing Contamination

Summary

In this Illumina community video, Imani Bethel and Tim Gilmartin discuss the importance of minimizing contamination in next-generation sequencing applications that require DNA amplification through PCR. They highlight best practices such as establishing separate pre- and post-PCR areas, using dedicated equipment and PPE, and maintaining a unidirectional workflow. The video also emphasizes proper pipetting techniques, routine cleaning with bleach solutions, and the use of no-template controls to ensure robust and reproducible data.

Takeaways

• 🔬 **PCR Contamination Risk**: Next-generation sequencing applications often require DNA amplification via PCR, which can introduce contamination risks.
• 🏠 **Lab Layout**: Establish separate pre-PCR and post-PCR areas to prevent cross-contamination, ideally in different rooms with physical separation.
• 🛠️ **Dedicated Equipment**: Use separate equipment for pre- and post-PCR processes to avoid cross-contamination.
• 🧼 **Strict Cleaning**: Maintain a rigorous cleaning schedule for all lab areas and equipment to minimize the risk of contamination.
• 🧤 **Personal Protective Equipment (PPE)**: Use different PPE for pre- and post-PCR areas and change gloves frequently to prevent contamination.
• 📚 **Unidirectional Workflow**: Process samples in a single direction (pre-PCR to post-PCR) to reduce the chance of contamination.
• 💧 **Proper Pipetting Technique**: Ensure correct pipetting technique to avoid sample-to-sample contamination due to aerosols or liquid transfer.
• 🧪 **No Template Control (NTC)**: Include NTCs in experiments to identify and monitor contamination in samples, reagents, or the lab environment.
• 🧴 **Aerosol-Resistant Tips**: Use aerosol and liquid-resistant pipette tips to prevent contamination from aerosols.
• 🧹 **Routine Cleaning**: Perform routine cleaning with bleach solutions to sanitize lab surfaces and equipment, and make fresh solutions daily.

Q & A

What is the primary concern with DNA amplification in next-generation sequencing applications?

The primary concern with DNA amplification, such as through PCR, is the potential for contamination, which can lead to increased background noise in data and even false discoveries.

How can physical separation of pre- and post-PCR areas help in preventing contamination?

Physical separation of pre- and post-PCR areas can prevent contamination by ensuring that amplified material does not re-enter the pre-PCR area, thus reducing the risk of sample cross-contamination.

What are the recommended measures for maintaining a clean pre-PCR area?

Maintaining a clean pre-PCR area involves maintaining positive air pressure, adding pass-through airlocks, and using separate equipment and personal protective equipment (PPE) for pre- and post-PCR areas.

Why is it important to process samples in a unidirectional workflow from pre-PCR to post-PCR?

A unidirectional workflow helps prevent potential sources of contamination from returning to the pre-PCR area, thus reducing the risk of cross-contamination.

What should be done when a lab member must return to the pre-PCR area after working in the post-PCR area?

If a lab member must return to the pre-PCR area after working in the post-PCR area, they should put on fresh PPE and thoroughly clean all areas in the pre-PCR area.

How can proper pipetting technique help in avoiding contamination?

Proper pipetting technique, such as ensuring the tip is securely seated, holding the pipettor vertically, and aspirating the correct volume, can help avoid sample-to-sample contamination due to aerosols.

What is the role of aerosol and liquid resistant pipette tips in preventing contamination?

Aerosol and liquid resistant pipette tips, often with self-sealing filters, prevent liquids or aerosols from contacting the pipettor, thus reducing the risk of contamination.

Why is it recommended to change pipette tips between each sample when working with Illumina library preparation kits?

Changing pipette tips between each sample prevents cross-contamination and ensures that each sample is processed with clean, uncontaminated equipment.

What is the significance of including a no-template control (NTC) in experiments involving amplification?

Including an NTC helps identify contamination in samples, reagents, or the laboratory environment by checking for amplified products in the absence of a template.

How often should routine cleaning with bleach solution be performed to prevent contamination?

Routine cleaning with a bleach solution is recommended on a daily and weekly basis, with thorough cleaning occurring weekly.

What are the common areas that should be targeted for cleaning to prevent contamination in the lab?

Common areas to target for cleaning include pipette tips, bench tops, equipment surfaces, control panels for lab equipment, handles for refrigerators and freezers, and any other surfaces that are frequently touched during lab operations.