Multi-drug-resistant Neisseria gonorrhoeae infection is a significant public health risk. Rapidly detecting N. gonorrhoeae and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of N. gonorrhoeae DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire N. gonorrhoeae genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01–0.1 %) to 76 % (42–82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112–720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.

中文翻译：

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靶标富集可通过纳米孔测序直接从临​​床样本中提高对淋病奈瑟菌和抗菌素耐药性决定因素的独立培养检测

多重耐药淋病奈瑟菌感染是一个重大的公共卫生风险。通过尿液宏基因组测序快速检测淋病奈瑟菌和抗菌素耐药性 (AMR) 决定因素是可能的，尽管高水平的宿主 DNA 和污染物种的过度生长阻碍了测序并限制了淋病奈瑟菌基因组覆盖范围。我们使用定制的 SureSelect panel 对核酸扩增测试阳性尿样和培养阳性尿道拭子进行了纳米孔测序，无论是否有基于探针的目标富集，以研究淋病奈瑟菌DNA 的选择性富集是否可以改善对两种物种的检测AMR 决定因素。探针被设计为覆盖整个淋病奈瑟菌基因组，十倍富集的探针覆盖选定的 AMR 决定簇。在样本子集中测试了多重分析。富集后，所有样本中被归类为淋病奈瑟菌的序列碱基比例均有所增加，中位数 (IQR) 为 0.05% (0.01–0.1%) 至 76% (42–82%)，基因组倍数中位数也相应提高覆盖365次（112-720）。 13/15 (87 %) 的样品实现了稳健的 AMR 决定簇检测所需的超过 20 倍的覆盖率，而未经富集的样品为 2/15 (13 %)。复用在一起的四个样本也实现了超过 20 倍的基因组覆盖率。 AMR 决定簇的覆盖范围足以预测染色体基因变化（如果存在）所赋予的耐药性，并且基因组覆盖范围也使得系统发育关系得以重建。当对直接从尿液或尿道拭子提取的 DNA 进行测序时，基于探针的靶标富集可以提高淋病奈瑟菌基因组覆盖度，从而检测 AMR 决定簇。此外，富集之前的多重分析为 AMR 检测提供了足够的基因组覆盖度，并降低了与该方法相关的成本。