Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria
Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria
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细菌拉曼激活细胞喷射和测序的合理优化
DOI：
10.1021/acs.analchem.9b05345
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发表时间：
2020-06-16
影响因子：
7.4
通讯作者：
Xu, Jian
中科院分区：
文献类型：
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作者：
Su, Xiaolu;Gong, Yanhai;Xu, Jian
在拉曼激活细胞弹射和测序（RACE-Seq）中，单个RACE后细胞的鸟枪测序的成功率和序列覆盖率通常较低。在这里，我们定量评估了细胞裂解条件、核酸扩增条件和拉曼测量参数对RACE-Seq性能的影响。在拉曼信号采集过程中激光能量输入的变化，而不是碱性裂解物裂解的持续时间、温度或在干燥或含水条件下的测量，对多重置换扩增（MDA）的成功至关重要。事实上，激光辐照与MDA产品质量密切相关。然而，在MDA之前引入油，通过减轻拉曼照射的这种负面影响，提高了RACE后大肠杆菌细胞的基因组覆盖率，
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 Escherichia coli cells from