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Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria.

In Raman-activated Cell Ejection and Sequencing (RACE-Seq), success rate and sequence coverage have generally been low for shotgun sequencing of individual post-RACE cells. Here we quantitatively evaluated the influence of cell lysis condition, nucleic acid amplification condition, and parameters of Raman measurement on RACE-Seq performance. Variations in laser energy input during Raman signal acquisition, but not duration of alkaline lysate lysis, temperature, or measurement under dry or aqueous conditions, are vital to the success of multiple displacement amplification (MDA). In fact, laser irradiation is reversely linked to MDA product quality. However, introduction of oils prior to MDA, by mitigating such negative effects of Raman irradiation, elevates genome coverage of post-RACE E. coli cells from <20% to ~50%, while greatly improves the success rate of RACE-Seq for soil microbiota. Our findings provide a practical solution for enhancing RACE-Seq performance, and pinpoint protection of cells from laser irradiation as priority in method development.