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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 被引量:1
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作者 陈志森 黄子夏 +1 位作者 陈军 柯才焕 《厦门大学学报(自然科学版)》 CAS CSCD 北大核心 2012年第4期774-781,共8页
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10nt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(P... 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10nt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转录组文库的DNA浓度,3种方法的数值均一化并加权平均后,按照预定比例混合.为评估文库质量,先采用传统的Sanger测序测定了混合文库的192条序列,发现191条序列具有符合Roche/454高通量测序要求的AB格式,即一端为454A序列,另一端为454B序列;其中189条序列能分辨出其带有的多重标签;插入的平均长度为348bp.然后通过Roche/454的滴定测序获得了34642条序列,其中32872条序列(94.9%)能分辨出其带有的多重标签,能够精确地分配到12个转录组,并且利用滴定测序结果计算出每个转录组文库的真实比例,对定量PCR、Qubit和NanoDrop-1000进行了评估,结果表明定量PCR是相对准确的定量方法.以上的评估表明所建立的转录组文库构建方法是稳定可靠的,可以广泛应用于转录组学研究. 展开更多
关键词 转录组 roche/454高通量测序 多重标签 PCR抑制效应
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Bacterial Community Structure and Diversity of Closely Located Coastal Areas
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作者 Md. Nurul Haider Masahiko Nishimura Kazuhiro Kogure 《Open Journal of Marine Science》 2016年第3期423-439,共17页
Bacterial community structure and diversity of two closely located stations are usually considered similar which can be verified by more intensive investigations using relatively large amount of datasets from the next... Bacterial community structure and diversity of two closely located stations are usually considered similar which can be verified by more intensive investigations using relatively large amount of datasets from the next generation sequencer. This study was conducted to assess the bacterial community structure and diversity between two closely located coastal stations, the port side and the sea side of the Oarai, Ibaraki, Japan from March 2013 to July 2014 using 454 GS Junior sequencer. Two stations underwent similar changes in physicochemical properties but the community structure and diversity was different. The Proteobacteria (the class Alphaproteobacteria, followed by the Gammaproteobacteria) and the Bacteroidetes (the class Flavobacteriia) were two abundant phyla in both the stations. But, the Flavobacteriia was more abundant in the port side, contributed about 26% to 48%, compared to the sea side (about 12% to 39%). Conversely, the relative abundance of the Gammaproteobacteria was higher on the sea side, about 10% to 17%, compared to the port side (about 4% to 12%). Among others, the phyla Cyanobacteria, Deferribacteres, Verrucomicrobia and the class Betaproteobacteria were also relatively abundant at the sea side. Because of their dominancy, the class Flavobacteriia and Alphaproteobacteria were further analysed at a lower phylogenetic level and marked differences were observed between the stations. Bacterial biodiversity in terms of the species richness (Chao index) and evenness (inverse Simpson) indicated higher patterns of diversity in the sea side area compared to the port side. Non-metric Multidimensional Scaling fitting with the environmental features (metaMDS), redundancy analysis (RDA) and Bray-Curtis clustering analysis also showed marked differences in bacterial community structure and diversity between the stations. However, some OTUs were commonly found in both the stations in all the sampling periods. So, the bacterial community structure and diversity of the coastal areas are distinguishable even between two closely located sampling points. 展开更多
关键词 Coastal Bacteria Community Structure DIVERSITY High-Throughput Sequencing roche 454
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基于蓝藻全基因组原始数据的转座元件挖掘及组成分析
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作者 肖鹏 李仁辉 《遗传》 CAS CSCD 北大核心 2011年第6期654-660,共7页
二代测序技术及全基因组多样性比较是现代生物学及信息科学研究的热点,对基因组中转座元件(Transposable element)的分析已成为基因组比较分析的重要组成部分。目前对于转座元件的种类、数量和组成的挖掘和分析一般是基于完全拼接后的... 二代测序技术及全基因组多样性比较是现代生物学及信息科学研究的热点,对基因组中转座元件(Transposable element)的分析已成为基因组比较分析的重要组成部分。目前对于转座元件的种类、数量和组成的挖掘和分析一般是基于完全拼接后的全基因组序列,对在此之前的海量短片段序列后期处理及拼接仍是目前基因组研究的盲点,以转座元件为主的重复序列在拼接过程中也存在着不可避免的拼接误差或丢失,给转座元件系统的分析带来不确定。文章旨在建立一套分析流程,对铜绿微囊藻NIES 843全基因组构建的罗氏(Roche)公司454测序随机模拟原始数据集的转座元件(主要类型为插入序列:Insert sequence,IS)组成进行分析,结果表明,采用对核酸探针扫描后备选序列分成3组,并分设氨基酸检测阈值的方案分析得到的结果较为可靠,结果显示铜绿微囊藻NIES843的蓝藻转座元件占基因组比例的10.38%,归属于14个IS家族,66个IS亚家族。与之前基于完整拼接基因组数据的两套不同分析流程得到的结果相比,在丰度及家族/亚家族组成上无显著差异,在转座元件序列水平上也显示了高比例的相似性序列重叠,证实了本研究流程在基于高通量测序原始数据的转座元件分析方面具可靠性及实用性。 展开更多
关键词 蓝藻基因组 插入序列 IS家族 转座元件 roche454测序原始数据
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