A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.
A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.
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DOI：
10.1186/1471-2164-15-828
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发表时间：
2014-09-30
期刊：
影响因子：
4.4
通讯作者：
Cottam EM
中科院分区：
文献类型：
--
作者：
Logan G;Freimanis GL;King DJ;Valdazo-González B;Bachanek-Bankowska K;Sanderson ND;Knowles NJ;King DP;Cottam EM
下一代测序(NGS)通过提供新的方法在诊断环境中对各种人类和兽医病原体进行全基因组测序(WGS)，正在给分子流行病学带来革命性的变化。以前的测序方案受到偏差的影响，例如在PCR扩增和细胞培养过程中遇到的偏差，或者受到对大量起始材料的需求的限制。我们在这里描述了一种简单而健壮的方法，用于在Illumina MiSeq上生成全基因组序列。该方案专用于口蹄疫病毒(FMDV)或其他多腺化RNA病毒，避免了使用聚合酶链式反应和大量初始模板的要求。该方案使用2001年英国疫情的5个口蹄疫病毒阳性临床样本以及来自所有7个血清型的一组代表性病毒成功验证。此外，该方案被成功地用于恢复94%的口蹄疫病毒基因组，这些基因组以前被鉴定为细胞培养阴性。对其他三种非口蹄疫病毒多腺化RNA病毒(EMCV、ERAV、VESV)的基因组序列也进行了轻微的修改。我们计算出口蹄疫病毒O的最小覆盖深度需要22个读数才能产生准确的共识序列。这在5个口蹄疫病毒/O/UKG分离物和O型口蹄疫病毒血清型小组中实现了，但5‘基因组末端和紧邻Poly(C)区的区域除外。我们开发了一种通用的口蹄疫病毒和其他多腺化RNA病毒的WGS方法。这种方法从有限的起始材料中成功地进行了工作，并且消除了对基因组特异性PCR扩增的要求。该协议有可能在常规的高通量诊断环境中产生共识水平的序列。本文的在线版本(DOI：10.1186/1471-2164-15-828)包含补充材料，可供授权用户使用。
Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region. We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment. The online version of this article (doi:10.1186/1471-2164-15-828) contains supplementary material, which is available to authorized users.
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DOI：
10.1093/bioinformatics/btq033
发表时间：
2010-03-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Quinlan AR;Hall IM
通讯作者：
Hall IM
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
DOI：
10.2460/javma.2002.220.1636
发表时间：
2002-06-01
期刊：
JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION
影响因子：
--
作者：
Callahan, JD;Brown, F;Nelson, WM
通讯作者：
Nelson, WM
影响因子：
3.7
作者：
Daly GM;Bexfield N;Heaney J;Stubbs S;Mayer AP;Palser A;Kellam P;Drou N;Caccamo M;Tiley L;Alexander GJ;Bernal W;Heeney JL
通讯作者：
Heeney JL
影响因子：
9.5
作者：
Milne, Iain;Stephen, Gordon;Marshall, David
通讯作者：
Marshall, David