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[1]陈志森,黄子夏,陈军*,等.用于Roche/454高通量测序的12个多重标签转录组文库的构建[J].厦门大学学报(自然科学版),2012,51(4):774.
 CHEN Zhi sen,HUANG Zi xia,CHEN Jun*,et al.Construction of 12 Multiplex Transcriptome Libraries for Roche/454 Pyroseqeuencing Platform[J].Journal of Xiamen University(Natural Science),2012,51(4):774.
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用于Roche/454高通量测序的12个多重标签转录组文库的构建(PDF)
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《厦门大学学报(自然科学版)》[ISSN:0438-0479/CN:35-1070/N]

卷:
51卷
期数:
2012年第4期
页码:
774
栏目:
研究论文
出版日期:
2012-07-15

文章信息/Info

Title:
Construction of 12 Multiplex Transcriptome Libraries for Roche/454 Pyroseqeuencing Platform
作者:
陈志森黄子夏陈军*柯才焕
厦门大学海洋与地球学院,福建 厦门 361005
Author(s):
CHEN ZhisenHUANG ZixiaCHEN Jun*KE Caihuan
College of Ocean and Earth Sciences,Xiamen University,Xiamen 361005,China
关键词:
转录组Roche/454高通量测序多重标签PCR抑制效应
Keywords:
transcriptomeRoche/454 pyroseqeuencingmultiplex identifier (MID)PCR suppression
分类号:
Q 356.1
文献标志码:
-
摘要:
利用第二代测序技术测定海洋生物转录组已成为揭示海洋生物分子生物学机制的重要工具.以长度为10 nt的不同多重标签(multiplex identifier)进行区分,构建了杂色鲍(Haliotis diversicolor)、僧帽牡蛎(Saccostrea cucullata)和亚心形扁藻(Platymonas subcordiformis)3种海洋生物的12个多重标签转录组文库.以合适的比例对各文库进行混合,利用定量PCR、Qubit和NanoDrop1000等3种DNA浓度测定方法测定每个多重标签
Abstract:
Nextgeneration sequencing technologies have become an important tool to promote research on molecular biologic mechanism of marine organisms. In this study,12 transcriptomice libraries,each labeled by a specific multiplex identifier (MID) of 10 nucleotide,were constructed from three marine species:small abalone Haliotis diversicolor,Sengmao oyster Saccostrea cucullata and unicell green algae Platymonas subcordiformis.To make an appropriate mixture ratio,DNA concentration of each library was determined by qPCR,Qubit and NanoDrop1000 methods.The normalized and weighted average concentration numbers were used to pool libraries to a setting ratio.To evaluate the quality of the libraries,traditional Sanger sequencing was performed to sequence 192 cDNA fragments.Among them,191 fragments had the required AB format on Roche/454 platform,e.g.,454A sequence was located on one end of the cDNA fragment and 454B sequence was located on the other end.And MID barcodes could be identified from 189 cDNA fragments. The average length of the cDNA fragments was 348 bp.To further evaluate the libraries, a titrimetric sequencing was performed on a Roche/454 GSFLXTitanium platform and 34 642 sequences were generated.MID barcodes could be identified from 32 872 sequences (94.9%) and each of them could be precisely assigned to one of the 12 cDNA libraries.The real proportions of each MID calculated from titrimetric sequences were used to evaluate the three DNA concentration determined methods,playing an important role in library pooling.Results showed that the qPCR method could be the most credible one.As a conclusion, the transcriptomice library construction procedure described here was very stable and reliable,and it could be widely applied on transcriptomics research.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:20110816基金项目:国家重点基础研究发展计划(973)项目(2010CB126403);国家自然科学基金项目(40976093);福建省青年科技人才创新项目(2008F3098)*通信作者:chenjun@xmu.edu.cn
更新日期/Last Update: 2012-07-15