Life in the Lab: A DNA sequencing pipeline

Introduction

This tutorial provides a comprehensive overview of the DNA sequencing pipeline at the Wellcome Trust Sanger Institute. It outlines the key steps involved in processing DNA samples, from initial quality checks to data release for researchers. Understanding this process is essential for anyone interested in laboratory skills and DNA research.

Step 1: Sample Reception and Quality Control

• Receive Samples: DNA samples arrive in 96 well plates, already extracted and purified from cell tissue or blood.
• Check Quality: Assess the quality of DNA to ensure it consists of long strings (genomic DNA).
• Use of Mosquito Robot:
  • Employ the Mosquito robot to pipette nanoliters of DNA for minimal sample usage.
  • Mix the DNA with a fluorescent dye that glows only in the presence of DNA.
• Analyze Fluorescence: Measure the level of fluorescence to determine DNA concentration.

Step 2: Library Preparation

• Library Construction: Conduct library preparation in the pre-PCR lab using Bravo robots.
• Attach Adaptors:
  • Pipette solutions containing adaptors, which are short DNA fragments.
  • Adaptors serve two purposes:
    • Allow samples to attach to the flow cell.
    • Contain tags for identifying DNA fragments during sequencing.
• Barcode Tracking: Utilize barcodes on each plate and a Laboratory Information Management System (LIMS) to track samples throughout the preparation process.

Step 3: PCR Amplification

• Transfer Samples: Move samples from the robot to a designated clean bench area, organized with tape and labels.
• Prevent Cross-Contamination:
  • Seal plates tightly to avoid evaporation during high-temperature PCR cycles.
  • Conduct PCR amplification in a separate lab to prevent contamination.
• Use of Color-Coded Lab Coats: Different lab coat colors help distinguish personnel and minimize contamination risks.

Step 4: Sequencing Preparation

• Remove Seal Carefully:
  • Manually remove seals from PCR plates to avoid sample splashes and contamination. This step cannot be performed by robots.
• Load Samples onto Flow Cell: Place prepared samples onto the flow cell to prepare for sequencing.
• Initiate Sequencing Run: Start the sequencer and monitor data quality throughout the run.

Step 5: Data Management

• Data Transfer: Once sequencing is complete, transfer the data to a data center equipped with a powerful computer cluster.
• Final Quality Check: Verify that sufficient and high-quality data has been produced.
• Data Access for Researchers: Once confirmed, researchers can access the sequencing data through the data cluster.

Conclusion

The DNA sequencing pipeline involves meticulous steps that ensure the integrity and quality of samples throughout the process. From quality control to data management, each stage is critical for successful DNA analysis. For those interested in laboratory skills, understanding this pipeline offers valuable insights and practical applications in the field of genetics and molecular biology. Consider exploring additional resources or hands-on experiences to deepen your knowledge of DNA sequencing techniques.