ABI3130xl全自动测序仪规模化测序的影响因素研究

Analysis of several factors affecting large-scale sequencing using ABI3130xl automatic genetic analyzer

【目的】提高利用ABI3130xl全自动测序仪对小麦与条锈菌互作的cDNA文库进行大规模测序的成功率,降低成本。【方法】将BigDye(BigDye(Teminator V3.1 Cycle Sequencing RR-100)分别稀释16,24和32倍设计测序的PCR反应体系,分析了EDTA溶液(125 mmol/L,pH 7.0)和醋酸钠溶液(3 mol/L,pH 5.2)以及不同模板类型等相关因素对测序结果的影响。【结果】BigDye稀释16倍的PCR反应体系的测序结果易出现染料峰,影响碱基正确读值;稀释32倍体系的测序结果中可用序列短,成功率低;稀释24倍体系测序结果的可用序列长且准确率高,为最佳反应体系。EDTA溶液和醋酸钠溶液可提高DNA纯化的质量,改善测序效果。以质粒DNA为模板测序较PCR产物直接测序效果好,通过控制模板质量可以提高测序成功率。【结论】利用优化体系共测得15 000条ESTs序列,其中有高读长序列13 080条,占87.2%,表明该优化体系简单有效,且可有效节约成本。

[Objective] The study is to develop the rate of successful sequencing and reduce the cost when operating large-scale sequencing to cDNA library from interaction between wheat and Puccinia striiformis f. sp. tritici using ABI3130xl automatic genetic analyzer. [Method] Several factors,including se- quencing PCR reaction systems using BigDye （BigDye） Teminator V3.1 Cycle Sequencing RR-100） diluted 16,24 and 32 times respectively,EDTA solution （125 mmol/L,pH7.0） and NaAc solution （3 mol/L,pH 5.2） ,as well as different template types,which might affect sequencing results,were analyzed. [Result] The result showed that PCR reaction system with BigDye diluted 16 times led to high noisy signals which produced bad results in base pairs reading,and the one with BigDye diluted 32 times led to short valid sequences and low rate of successful sequencing, while the one with BigDye diluted 24 times acquired long valid sequences with high accuracy,so we chose the one with BigDye diluted 24 times as the best reaction system;EDTA solution and NaAc solution can enhance the quality of DNA purification,which can develop the quality of sequencing results Plasmid DNA as template is better than PCR products for sequencing, and good template can develop the rate of successful sequencing. [Conclusion] 15 000 ESTs were sequenced using the optimized system in the research,of which 13 080 sequences were High LOR （Length of Read）. It showed that the optimized system is simple and can reduce cost efficiently.