Background

Mycoplasma genitalium resistance to antibiotic treatments is increasing, with very limited treatment alternatives on the horizon. Surveillance via sequencing of multiple M. genitalium loci would allow: monitoring of known antibiotic resistance mutations, associations between resistance/treatment failure and specific mutations, and strain typing for epidemiological purposes. In this study we assessed the performance of a custom amplicon sequencing approach, which negates the cost of library preparation for next generation sequencing.

Methods

Fifty-two M. genitalium positive samples (cervical, vaginal, anal and rectal swabs, and urine) were used. Three regions associated with M. genitalium antibiotic resistance (23S rRNA, parC and gyrA genes) were targeted, in conjunction with a locus used for differentiation of sequence types in the mgpB gene, and findings compared to Sanger sequencing.

Results

Amplicon sequencing provided adequate sequence read coverage (>30×) for the majority of samples for 23S rRNA gene (96%) and mgpB (97%), parC (78%) and gyrA (75%). Single nucleotide polymorphisms (SNPs) were characterised in samples for 23S rRNA gene (94%), parC (56%) and gyrA (4%). Unlike Sanger sequencing, mixed mutations could be identified by the amplicon sequencing method, and ratios of mutation types determined. All results, with one exception, were concordant to Sanger sequence results. Sequence diversity in the mgpB region was represented by 15 sequence types, 4 being observed in multiple samples.

Conclusions

We have demonstrated the utility of this custom amplicon sequencing approach for generating highly informative datasets with the capacity to identify and determine ratios of mixed sequences. The use of this customisable amplicon sequencing method enables cost effective, scalable amplicon sequencing of multiple target regions of interest in M. genitalium.

中文翻译：

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一种定制扩增子测序方法，可检测生殖器支原体中与耐药相关的突变和序列类型

背景

生殖支原体对抗生素治疗的耐药性正在增加，即将出现的治疗选择非常有限。通过对多个生殖器支原体基因座进行测序的监测可以：监测已知的抗生素耐药性突变，耐药性/治疗失败与特定突变之间的关联以及用于流行病学目的的菌株类型。在这项研究中，我们评估了定制扩增子测序方法的性能，该方法消除了用于下一代测序的文库制备成本。

方法

使用五十二个生殖器支原体阳性样品（宫颈，阴道，肛门和直肠拭子以及尿液）。靶向与生殖器分枝杆菌抗生素抗性相关的三个区域（23S rRNA，parC和gyrA基因），以及用于区分mgpB基因序列类型的基因座，并将其发现与Sanger测序进行比较。

结果

扩增子测序为23S rRNA基因（96％）和mgpB（97％），parC（78％）和gyrA（75％）的大多数样品提供了足够的序列读取覆盖率（> 30x）。在23S rRNA基因（94％），parC（56％）和gyrA（4％）的样品中鉴定了单核苷酸多态性（SNP）。与Sanger测序不同，可以通过扩增子测序方法鉴定混合突变，并确定突变类型的比率。除一个例外，所有结果均与Sanger序列结果一致。mgpB区的序列多样性由15种序列类型表示，在多个样品中观察到4种。

结论

我们已经证明了这种定制扩增子测序方法的实用性，该工具可用于生成高度信息化的数据集，并具有识别和确定混合序列比率的能力。这种可定制的扩增子测序方法的使用使得能够对生殖器支原体中的多个目标靶区域进行具有成本效益的，可扩展的扩增子测序。