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Published on: August 7, 2021
Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria.
Xiaolu Su1,2, Yanhai Gong1,2, Honglei Gou1,2
1Single-Cell Center, CAS Key Laboratory of Biofuels, Shandong Key Laboratory of Energy Genetics and Shandong Institute of Energy Research, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China.
Protecting cells from laser irradiation during Raman-activated cell ejection and sequencing (RACE-Seq) is key to improving success. Adding oils before amplification significantly boosts genome coverage and success rates for sequencing microbial communities.
Area of Science:
- Molecular Biology
- Genomics
- Microbiology
Background:
- Raman-activated cell ejection and sequencing (RACE-Seq) is a method for single-cell genomics.
- Current RACE-Seq protocols suffer from low success rates and poor genome coverage for individual cells.
Purpose of the Study:
- To quantitatively assess factors influencing RACE-Seq performance.
- To identify key parameters for optimizing single-cell genome sequencing after cell ejection.
Main Methods:
- Evaluated effects of cell lysis conditions, nucleic acid amplification parameters, and Raman measurement settings.
- Investigated the impact of laser energy, lysis duration, temperature, and measurement medium (dry vs. aqueous).
- Assessed the role of oil introduction prior to multiple displacement amplification (MDA).
Main Results:
- Laser energy input during Raman measurement critically affects multiple displacement amplification (MDA) success, with higher energy negatively impacting product quality.
- Lysis duration, temperature, and measurement conditions (dry/aqueous) had minimal impact on MDA.
- Introducing oils before MDA mitigated negative effects of laser irradiation, increasing genome coverage from <20% to ~50% for *Escherichia coli* and improving success rates for soil microbiota.
Conclusions:
- Protecting cells from laser-induced damage during Raman measurement is crucial for enhancing RACE-Seq performance.
- The addition of oils prior to MDA offers a practical strategy to improve genome coverage and success rates in RACE-Seq.
- Optimizing laser parameters and implementing protective measures are priorities for future RACE-Seq method development.
