背景:下一代测序(NGS)通过提供新的方法在各种人类和兽医病原体的诊断环境中进行全基因组测序(WGS),正在彻底改变分子流行病学。以前的测序方案受到偏见,例如在PCR扩增和细胞培养过程中遇到的偏见,或者由于需要大量的起始材料而受到限制。我们在这里描述了在IlluminaMiSeq上生成全基因组序列的简单而强大的方法。该方案对口蹄疫病毒(FMDV)或其他聚腺苷酸化RNA病毒具有特异性,并且避免了PCR的使用和对大量初始模板的要求。
结果:使用来自2001年英国流行病的五个FMDV阳性临床样本成功验证了该方案,以及一组来自所有七个血清型的代表性病毒。此外,该方案已成功用于回收先前被鉴定为细胞培养阴性的FMDV基因组的94%.来自其他三种非FMDV聚腺苷酸化RNA病毒的基因组序列(EMCV,ERAV,VESV)也通过少量的协议修改获得。我们计算出,为FMDVO产生准确的共有序列需要22个读数的最小覆盖深度。这在5个FMDV/O/UKG分离株和血清型组中的O型FMDV中实现,除了5'基因组末端和紧接poly(C)区域的区域。
结论:我们已经开发了一种针对FMDV和其他聚腺苷酸化RNA病毒的通用WGS方法。该方法成功地从有限数量的起始材料起作用,并且消除了对基因组特异性PCR扩增的需要。该方案具有在常规高通量诊断环境中产生共有水平序列的潜力。
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.