基于WGP物理作图法的虾夷扇贝Fosmid克隆混池策略及解码率研究  

Investigation of Fosmid Clone Pooling Strategy and Decoding Rate for WGP-Based Physical Mapping in Yesso Scallop

作　　者：窦怀乾 李仰平[1] 吕佳[1,2] 窦锦壮 李语丽[1,2] 王师 DOU Huai-Qian;LI Yang-Ping;LV Jia;DOU Jin-Zhuang;LI Yu-Li;WANG Shi(Laboratory of Marine Genetics and Breeding,Ministry of Education Key,College of Marine Life Sciences,Ocean University of China,Qingdao 266003,China;Laboratory for Marine Biology and Biotechnology,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266237,China)

机构地区：[1]中国海洋大学海洋生物遗传学与育种教育部重点实验室,山东青岛266003 [2]海洋生物学与技术功能实验室青岛海洋科学与技术国家实验室,山东青岛266237

出　　处：《中国海洋大学学报（自然科学版）》2019年第2期44-51,共8页Periodical of Ocean University of China

基　　金：中央高校基本科研业务费专项(201564009;201762001);青岛海洋科学与技术国家实验室"鳌山人才"计划项目(2015ASTP-ES02)资助~~

摘　　要：利用全基因组解析(Whole genome profiling,WGP)法进行物理图谱构建时,克隆混池策略及克隆解码率的高低是影响物理图谱构建效果的关键性因素。如何通过调控混合克隆数目,来平衡克隆解码率和测序的成本,是利用WGP法构建物理图谱的亟需解决的问题。本研究利用虾夷扇贝Fosmid文库的部分克隆,使用序列特异性标签的WGP方法,对虾夷扇贝物理图谱构建方法的混池策略及克隆解码率进行了初步研究。通过计算机分别模拟了384 N(N=1,2…24)孔培养板内的克隆混合,计算出了对应混合尺度下BsaXI和FspEI两种酶的克隆解码率。以该模拟数据作为参照,最终分别以4、8、12、16张384孔培养板内的克隆混合构建了克隆超级池;并利用2b-RAD技术构建了基于BsaXI和FspEI两种限制性内切酶的测序文库。通过对酶切标签数据的分析,成功实现了混合池内的克隆解码,且在利用两种酶对应的酶切标签联合解码后,联合解码率达到84%以上。本研究最终确定了由8张384孔培养板混合的混池策略及利用BsaXI和FspEI两种酶切标签进行联合克隆解码,为后期利用WGP方法构建虾夷扇贝物理图谱提供了参考方案。The clone pooling strategy and clone decoding rate are key factors that affect the efficiency of whole genome profiling （WGP）-based physical mapping approach. How to balance the clone decoding rate and the cost of sequencing by regulating the number of mixed clones is an issue that must be addressed when using the WGP method to construct a physical map. We used fosmid clones from a Yesso scallop fosmid library to explore the optimal clone pooling strategy and clone decoding rate for implementation of WGP-based physical mapping in Yesso scallop. By in silico analysis of a mixture of 384 N -well（ N =1,2…24）culture plates, the monoclonal decoding rate of Bsa XI and Fsp EI enzymes at the corresponding mixing scales were calculated. Based the simulation results, we chose to mix the real fosmid clones of Yesso scallop in 4, 8, 12, and 16 384-well bacteria culture plates and got the corresponding super clone pools. Using restriction enzymes Bsa XI and Fsp EI to build 2b-RAD libraries and sequencing the 2b-RADlibraries using the Illumina platform. Single clone was successfully decoded from the sequence data with the help of unique tags and Bsa XI tags has higher decoding rate than Fsp EI tags in every mixture level. When the tags from the two different enzymes were combined for clone decoding, the decoding rate exceeded 84%. Mixing eight 384-well bacteria culture plate and clone decoding based on Bs aXI and Fsp EI enzymes seem to be an optimal strategy. Our study provide the optimal clone pooling strategy for further implementation of WGP-based physical mapping in Yesso scallop.

关 键 词：虾夷扇贝 FOSMID文库 物理图谱 WGP 

分 类 号：Q75[生物学—分子生物学]