高通量测序H7N9禽流感病毒基因组模板制备方法的优化及应用

目的:建立一种较优的高通量测序H7N9禽流感病毒基因组模板制备方法,用于H7N9禽流感病毒基因组监测?方法:分别以6碱基随机引物反转录生成单链cDNA,合成双链cDNA;流感病毒通用反转录引物U12反转录生成单链cDNA,6碱基随机引物合成双链cDNA;U12引物反转录生成单链cDNA,通用流感病毒全基因扩增引物混合物生成双链cDNA制备高通量测序模板?构建测序文库?测序并分析数据,比较3种方法对高通量测序效率的影响,并与传统Sanger测序方法比较其测序的准确性,选择其中较优的方法用于高通量测序?结果:以U12引物反转录cDNA,通用流感病毒全基因扩增引物混合物生成双链cDNA制备的高通量测序模板,在测序族生成数?覆盖率?单核苷酸多态性及读长等方面均优于另外两种方法?结论:本研究建立的H7N9禽流感病毒基因组模板制备方法用于高通量测序具有准确性高,周期短,成本相对较低,可同时检测多个样本等特点,可用于大规模H7N9禽流感病毒基因组监测?

Optimization and application of methods of template preparation for high throughput sequencing avian influenza virus (H7N9) genome

Objective:To establish an optimal method of template preparation for sequencing avian influenza virus (H7N9) genome. Methods:Sequencing templates were prepared by using random hexamers primers to synthesize single strand cDNA and double strands cDNA;by using influenza virus specific-U12 primer to synthesize single strand cDNA and using random hexamers primers to obtain double strands cDNA;or by using influenza virus specific-U12 primer to synthesize single strand cDNA and using influenza virus gene-specific primer to obtain double strands cDNA. Sequencing library was constructed by using above template and sequencing. After analysis of the data,the results were compared with Sanger sequencing. The best method for template preparation was selected for high throughput sequencing. Results:The cluster pass filter,coverage,single nucleotide polymorphisms (SNPs),and median length were the best in using influenza virus specific-U12 primer to synthesize single strand cDNA and using influenza virus gene-specific primer to obtain double strands cDNA compared with other two methods. Conclusion:High accuracy,short turnaround times,relatively low cost and high throughput make this method becoming possible for surveillance of H7N9 viruses evolution.