简介

　　真菌基因组大小浮动范围相对较大，大多数是在40Mb以内，建议采用PacBio RS II平台测序4-8个SMRT Cell，获得3.2Gb-8Gb的数据，然后进行全基因组de novo拼接。以基因组大小为38Mb的真菌为例，测序8个SMRT Cell即可获得至少168X的覆盖度。

　　三代平台的测序错误来源于随机误差，因此可通过增加数据覆盖度显著降低错误率。根据PacBio官方提供的参考标准，当细菌/真菌基因组覆盖度大于80X时，碱基准确度已接近99.99999%（QV70），即相当于每一千万个碱基中约发生一个碱基错误。以上研究方案在碱基准确度、基因组覆盖度及组装结果方面均可获得较好结果。

技术优势

A、无GC偏好性，对于GC%正常菌株和GC%异常菌株，PacBio RS II平台均可获得高质量测序数据；

B、可获得0 gap基因组完成图，1个contig＝1条染色体；

C、对于包含质粒的菌株，不仅可获得完整的真菌基因组，还可同时获得完整的质粒基因组序列。




结果示例






应用案例

目前已经采用PacBio RS II平台开展的细菌和真菌物种有：嗜麦芽寡养单胞菌，金黄色葡萄球菌，大肠杆菌O104，乳酸菌，空肠弯曲菌，金黄色葡萄球菌，绿脓假单胞杆菌，梭状芽胞杆菌，耐辐射螺旋体菌，番茄溃疡病菌，淋病奈瑟氏菌，钩端螺旋体，甲基盐菌，赭色锁掷酵母，木拉科酵母，马克斯克鲁维酵母，葡萄穗霉，胶囊青霉，黄萎病菌，甘蔗鞭黑粉菌，艾滋病毒，茎菌属，肠道沙门氏菌，幽门螺杆菌，北里孢菌，嘴突脐孢，尖端赛多孢子菌，可杀死种传黄萎病菌等。下表2统计了应用PacBio RS II平台开展的微生物全基因组de novo项目（统计结果截止2015年11月）。




中文名	拉丁名	发表时间	刊物/会议	基因组大小
嗜麦芽寡养单胞菌	Stenotrophomonas maltophilia	2015	Antimicrob. Agents Chemother	4.5 Mb
金黄色葡萄球菌	Staphylococcus aureus	2015	BMC Genomics	2.87 Mb
大肠杆菌O104	Escherichia coli O104	2015	BMC Microbiology	4.99 Mb
乳酸菌	Lactobacillus hokkaidonensis	2015	BMC Genomics	2.28 Mb
空肠弯曲菌	Campylobacter jejuni	2015	Plos One	1.7 Mb
金黄色葡萄球菌	Staphylococcus aureus	2015	Genome Announcements	2.7 Mb
绿脓假单胞杆菌	Pseudomonas aeruginosa	2015	Genome Announcements	3.6 Mb
梭状芽胞杆菌	Clostridium sporogenes	2015	Genome Announcements	4.1 Mb
耐辐射螺旋体菌	Spirosoma radiotolerans,	2015	Genome Announcements	7 Mb
番茄溃疡病菌	Clavibacter michiganensis	2015	Genome Announcements	3.2 Mb
淋病奈瑟氏菌	Neisseria gonorrhoeae	2015	Genome Announcements	2.2 Mb
钩端螺旋体	Leptospira interrogans	2015	Genome Announcements	4.2 Mb
甲基盐菌	Methylohalobius crimeensis	2015	Genome Announcements	3.5 Mb
赭色锁掷酵母	Sporidiobolus salmonicolor	2015	Genome Announcements	20.5 Mb
木拉科酵母	Mrakia blollopis	2015	Genome Announcements	30.46 Mb
马克斯克鲁维酵母	Kluyveromyces marxianus	2015	Genome Announcements	10.89 Mb
葡萄穗霉	Stachybotrys chartarum	2015	Genome Announcements	41 Mb
胶囊青霉	Penicillium capsulatum	2015	Genome Announcements	34.37 Mb
黄萎病菌	Verticillium dahliae	2015	mBio	36 Mb
甘蔗鞭黑粉菌	Sporisorium scitamineum	2015	Plos One	19.9 Mb
艾滋病毒	HIV-1	2015	Conference proceeding	9 Kb
茎菌属	Caulobacter henricii	2014	Current Microbiology	3.9 Mb
肠道沙门氏菌	Salmonella enterica	2014	Genome Announcements	4.79 Mb
幽门螺杆菌	Helicobacter pylori	2014	Genome Announcements	1.6 Mb
北里孢菌	Kitasatospora cheerisanensis	2014	Genome Announcements	8 Mb
嘴突脐孢	Exserohilum rostratum	2014	J. Clin. Microbiol	33.8 Mb
尖端赛多孢子菌	Scedosporium apiospermum	2014	Genome Announcements	43 Mb
可杀死种传黄萎病菌	Verticillium tricorpus	2014	molecular plant-microbe interactions	36 Mb




参考文献

1、Abrams, A Jeanine., et al. (2015). Complete genome sequences of three Neisseria gonorrhoeae laboratory reference strains, determined using PacBio single-molecule real-time technology. Genome announcements.

2、Badouin, Hélène., et al. (2015). "Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae." Genetics.

3、Betancourt, Doris A., et al. (2015). "Genome sequence of Stachybotrys chartarum strain 51-11." Genome announcements.

4、basidiomycetous yeast." Genome announcements.  

5、Faino, Luigi., et al. (2015). "Single-Molecule Real-Time Sequencing Combined with Optical Mapping Yields Completely Finished Fungal Genome." MBio.      

6、Inokuma, Kentaro., et al. (2015). "Complete genome sequence of Kluyveromyces marxianus NBRC1777, a nonconventional thermotolerant yeast." Genome announcements.  

7、Kim, Myung Kyum., et al. (2015). "Complete genome sequence of Spirosoma radiotolerans, a Gamma-radiation-resistant bacterium isolated from rice field in South Korea." Journal of biotechnology.  

8、Li, Yi., et al. (2015). "Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis." Scientific reports.    

9、Lu, You., et al. (2015). "Complete genome sequence of Clavibacter michiganensis subsp. insidiosus R1-1 using PacBio single-molecule real-time technology." Genome announcements.