The one-week automated genome-wide optical pooled screen

Area of Science:

• Genomics
• High-content screening
• Bioinformatics

Background:

• Optical pooled screens (OPS) are powerful for large-scale genetic screens but are limited by complex, labor-intensive protocols for in situ sequencing and data analysis.
• Existing methods require significant manual effort and time, hindering the throughput and accessibility of OPS.

Purpose of the Study:

• To develop an automated platform, OttoSeq, that integrates fluid handling and data analysis to overcome the bottlenecks in optical pooled screens.
• To demonstrate the capability of OttoSeq in performing a genome-wide functional genomics screen efficiently.

Main Methods:

• Developed OttoSeq by combining the Otto2 fluid handling system with the Brieflow analysis pipeline for automated OPS.
• Executed a genome-wide cell painting screen using OttoSeq, involving the analysis of over 5 million cells across 21,732 gene knockout perturbations.
• Utilized automated data processing to interpret functional gene clusters.

Main Results:

• Successfully automated the entire OPS workflow, from cell perturbation to data interpretation.
• Completed a genome-wide cell painting screen in just eight days, a significant reduction in processing time.
• Analyzed over 5 million high-quality cells, providing 224 cells per gene knockout, and identified 320 distinct functional gene clusters.

Conclusions:

• OttoSeq represents a major advancement in automating optical pooled screens, making large-scale functional genomics more accessible and efficient.
• The platform's speed and capacity enable rapid exploration of cellular functions at a genome-wide scale.
• Automated OPS platforms like OttoSeq are crucial for accelerating biological discovery and understanding gene function.