摘要
      抗生素耐药性基因对21世纪的公共卫生和生态安全构成了严重威胁。然而，抗性组只占宏基因组含量的一小部分，这使得在各种环境中研究低丰度ARG变得困难。因此，需要一种高度敏感、准确和全面的方法来描述复杂宏基因组样本中的ARG图谱。在本研究中，我们建立了一种基于靶向扩增的高通量测序方法，该方法可以同时检测ARGs（n = 251）、可移动遗传元件基因（n = 8） 和金属抗性基因（n = 19） 在宏基因组中。将扩增子测序的性能与传统的宏基因组鸟枪测序（MetaSeq）进行了比较。总共设计了1421对引物，实现了目标基因的极高覆盖率。扩增子测序显著提高了靶ARGs的回收率（~9 × 104倍），具有更高的灵敏度和多样性，更少的成本和计算负担。此外，靶向富集可以对单核苷酸多态性（SNPs）进行深度扫描，本研究显示SNPs检测水平升高。我们进一步对48个环境样本（37个粪便、20个土壤和7个污水）和16个临床样本进行了这种方法。本研究中测试的所有样本都显示出靶基因的高度多样性和回收率。我们的研究结果表明，该方法可以应用于各种宏基因组样本，并作为ARGs监测和进化评估的有效工具。使用本研究中验证的富集方法获得抗性组，可以捕获低丰度抗性组，同时成本和时间更低，这可以极大地促进我们对局部和全球抗性组动力学的理解。
ABSTRACT
Antibiotic resistance genes (ARGs) pose a serious threat to public health and ecological security in the 21st century. However, the resistome only accounts for a tiny fraction of metagenomic content, which makes it difficult to investigate low-abundance ARGs in various environmental settings. Thus, a highly sensitive, accurate, and comprehensive method is needed to describe ARG profiles in complex metagenomic samples. In this study, we established a high-throughput sequencing method based on targeted amplification, which could simultaneously detect ARGs (n = 251), mobile genetic element genes (n = 8), and metal resistance genes (n = 19) in metagenomes. The performance of amplicon sequencing was compared with traditional metagenomic shotgun sequencing (MetaSeq). A total of 1421 primer pairs were designed, achieving extremely high coverage of target genes. The amplicon sequencing significantly improved the recovery of target ARGs (~9 × 104-fold), with higher sensitivity and diversity, less cost, and computation burden. Furthermore, targeted enrichment allows deep scanning of single nucleotide polymorphisms (SNPs), and elevated SNPs detection was shown in this study. We further performed this approach for 48 environmental samples (37 feces, 20 soils, and 7 sewage) and 16 clinical samples. All samples tested in this study showed high diversity and recovery of targeted genes. Our results demonstrated that the approach could be applied to various metagenomic samples and served as an efficient tool in the surveillance and evolution assessment of ARGs. Access to the resistome using the enrichment method validated in this study enabled the capture of low-abundance resistomes while being less costly and time-consuming, which can greatly advance our understanding of local and global resistome dynamics.

https://journals.asm.org/doi/full/10.1128/spectrum.02297-22