Brown Amanda Claire, Christiansen Mette T
Oxford Gene Technology, Oxford, UK.
Department of Microbiology and Immunology, Vet School, Cornell University, Ithaca, NY, USA.
Methods Mol Biol. 2019;2042:45-67. doi: 10.1007/978-1-4939-9694-0_6.
Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow growing, or obligate intracellular pathogens, such as Chlamydia trachomatis. However clinical samples typically contain too low levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a nonmutagenic approach, we can capture all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.
全基因组测序是一种强大的高分辨率工具，可用于生成有关细菌种群结构、系统地理学以及与抗菌药物耐药性相关突变的准确数据。直接从临床标本中对病原体基因组进行测序，而无需进行体外培养，在节省时间和人力方面很有吸引力，尤其是对于生长缓慢或专性胞内病原体，如沙眼衣原体。然而，临床样本中病原体核酸的含量通常过低，加上人类和天然微生物群DNA/RNA的含量相对较高，这使得直接测序成为一个不可行的选择。使用全基因组富集和深度测序相结合的方法，这已被证明是一种非诱变方法，我们可以捕获沙眼衣原体基因组中发现的所有已知变异。该方法是一种一致且灵敏的工具，能够直接从临床样本中快速对沙眼衣原体进行全基因组测序，并且有可能适用于其他具有相似克隆性质的病原体。
