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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。
Objective: To establish a better high-throughput sequencing H7N9 avian influenza virus genomic template preparation for H7N9 avian influenza virus genome monitoring. Methods: Single-stranded cDNA was reverse transcribed with a 6-bp random primer to generate double-stranded cDNA. The universal reverse transcription primer U12 of influenza virus was reverse transcribed into single-stranded cDNA and double-stranded cDNA with 6-mer random primers. U12 primer Reverse transcription to generate single-stranded cDNA, universal influenza virus whole-genome amplification primer mixture to generate double-stranded cDNA to prepare high-throughput sequencing template, build sequencing library, sequencing and analysis of data, compare the three methods of high-throughput sequencing efficiency, Compared with the traditional Sanger sequencing method, the accuracy of sequencing was compared and the best method was selected for high-throughput sequencing. Results: The high-throughput sequencing template of double-stranded cDNA was generated by U12 primer reverse transcriptase cDNA and universal influenza virus genome-wide amplification primer mixture. The sequence number, coverage, single nucleotide polymorphism Etc. are better than the other two methods. Conclusion: The H7N9 avian influenza virus genomic template preparation method established in this study is suitable for high-throughput sequencing with high accuracy, short cycle, low cost, simultaneous detection of multiple samples and other characteristics, and can be used for large-scale H7N9 bird flu virus Genome monitoring.