卡介苗美国株全基因组测序缺口修补及序列

Complete genome sequencing and sequence analysis of BCG Tice

摘要:

摘要:【目的】对卡介苗(Bacillus Calmette-Guerin，BCG)美国株(BCG Tice)进行基因组补缺口(补洞)工作，以得到它的基因组完整序列。【方法】首先对BCG Tice 进行高通量测序，使用SOAPdenovo 软件对得到的数据进行拼接。由于在高通量测序的过程中基因组某些区域测序覆盖度低，测序质量差会使测序结果经拼接后形成众多的重叠群( contig)，相邻的位置关系确定的contig 形成一个scaffold，contig 之间未测到的区域为缺口序列(gap)，在contig 末端设计引物进行PCR 扩增，得到连接相邻contig 的PCR 产物，对PCR 产物进行测序。通过优化PCR 引物设计策略，尝试不同的聚合酶进行聚合反应，调整PCR 反应条件并结合PCR 产物构建克隆测序等方法，补齐contig 之间的缺口序列。【结果】完成了BCG Tice 的全基因组测序，得到了它的基因组完整序列，序列已提交到美国国立生物技术信息中心(NCBI)的GenBank 数据库。【结论】BCG 属于高GC含量的革兰氏阳性细菌，其基因组GC含量高达65.65%。本文以BCG Tice 基因组补洞为例，对高GC含量基因组补缺口过程中遇到的问题与采取的策略给予概述，望给相关高GC 含量基因组的物种全基因组测序补缺口工作提供一些借鉴。

Abstract:

Abstract:［Objective］The objective of this study is to obtain the complete genome sequence of Bacillus Calmette-Guerin Tice (BCG Tice)，in order to provide more information about the molecular biology of BCG Tice and design more reasonable vaccines to prevent tuberculosis． ［Methods］ We assembled the data from high-throughput sequencing with SOAPdenovo software，with many contigs and scaffolds obtained． There are many sequence gaps and physical gaps remained as a result of regional low coverage and low quality． We designed primers at the end of contigs and performed PCR amplification in order to link these contigs and scaffolds． With various enzymes to perform PCR amplification，adjustment of PCR reaction conditions，and combined with clone construction to sequence，all the gaps were finished．［Results］We obtained the complete genome sequence of BCG Tice and submitted it to GenBank of National Center for Biotechnology Information (NCBI)．The genome of BCG Tice is 4334064 base pairs in length，with GC content 65. 65%．［Conclusion］The problems and strategies during the finishing step of BCG Tice sequencing are illuminated here，with the hope of affording some experience to those who are involved in the finishing step of genome sequencing． The microarray data were verified by our results.