Simplified large-scale Sanger genome sequencing for influenza A/H3N2 virus.
Simplified large-scale Sanger genome sequencing for influenza A/H3N2 virus.
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DOI：
10.1371/journal.pone.0064785
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Koay ES
中科院分区：
文献类型：
--
作者：
Lee HK;Tang JW;Kong DH;Koay ES
下一代测序技术的出现以及由此产生的测序成本的降低使得能够产生大量数据，包括产生病原体的全基因组序列。然而，流感病毒的小基因组大小证明了使用更常规的桑格测序技术是合理的，该技术目前在大多数诊断实验室中仍然更容易获得。我们提出了一种简化的基于Sanger的基因组测序方法，用于大规模测序A/H3 N2流感病毒。全基因组测序完成了19个逆转录-聚合酶链反应（RT-PCR）和39个测序反应。该方法分别在2009年至2011年期间收集的15份本地临床样本和15份培养分离株上进行了检测。15份天然临床样本记录的定量循环值范围为21.0 - 30.56，相当于2.4×103-1.4×106病毒拷贝/µL RNA提取物。所有PCR扩增产物不经纯化直接测序。值得注意的是，对于所有测试的样品，生成了高达700 bp的高质量测序数据。完成的序列总共覆盖408，810个核苷酸，每个基因组13，627个核苷酸，达到100%编码完整性。在所有产生的碱基中，平均89.49%是Phred质量值40（QV 40）碱基（代表每10，000个碱基约一个错误识别的准确度）或更高，平均93.46%是QV 30碱基（每1000个碱基一个错误识别）或更高。这种测序方案已被证明是具有成本效益的，并且在获得完整的流感基因组方面劳动密集度较低。通过适当使用适当设计的引物，产生的序列的恒定高质量赋予了将这种非纯化扩增子测序方法的应用扩展到其他基因测序测定的信心。
The advent of next-generation sequencing technologies and the resultant lower costs of sequencing have enabled production of massive amounts of data, including the generation of full genome sequences of pathogens. However, the small genome size of the influenza virus arguably justifies the use of the more conventional Sanger sequencing technology which is still currently more readily available in most diagnostic laboratories. We present a simplified Sanger-based genome sequencing method for sequencing the influenza A/H3N2 virus in a large-scale format. The entire genome sequencing was completed with 19 reverse transcription-polymerase chain reactions (RT-PCRs) and 39 sequencing reactions. This method was tested on 15 native clinical samples and 15 culture isolates, respectively, collected between 2009 and 2011. The 15 native clinical samples registered quantification cycle values ranging from 21.0 to 30.56, which were equivalent to 2.4×103–1.4×106 viral copies/µL of RNA extract. All the PCR-amplified products were sequenced directly without PCR product purification. Notably, high quality sequencing data up to 700 bp were generated for all the samples tested. The completed sequence covered 408,810 nucleotides in total, with 13,627 nucleotides per genome, attaining 100% coding completeness. Of all the bases produced, an average of 89.49% were Phred quality value 40 (QV40) bases (representing an accuracy of circa one miscall for every 10,000 bases) or higher, and an average of 93.46% were QV30 bases (one miscall every 1000 bases) or higher. This sequencing protocol has been shown to be cost-effective and less labor-intensive in obtaining full influenza genomes. The constant high quality of sequences generated imparts confidence in extending the application of this non-purified amplicon sequencing approach to other gene sequencing assays, with appropriate use of suitably designed primers.
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影响因子：
11.8
作者：
Chen, GW;Yang, CC;Shih, SR
通讯作者：
Shih, SR
影响因子：
4.8
作者：
Alvarez AC;Brunck ME;Boyd V;Lai R;Virtue E;Chen W;Bletchly C;Heine HG;Barnard R
通讯作者：
Barnard R
DOI：
10.1073/pnas.97.13.6974
发表时间：
2000-06-20
影响因子：
11.1
作者：
Bush, RM;Smith, CB;Fitch, WM
通讯作者：
Fitch, WM
影响因子：
46.9
作者：
Loman, Nicholas J.;Misra, Raju V.;Pallen, Mark J.
通讯作者：
Pallen, Mark J.
影响因子：
3
作者：
Archer J;Baillie G;Watson SJ;Kellam P;Rambaut A;Robertson DL
通讯作者：
Robertson DL