BACKGROUND Trypanosoma cruzi, the etiological agent of Chagas Disease, exhibits remarkable genetic diversity and is classified into different Discrete Typing Units (DTUs). Strain typing techniques are crucial for studying T. cruzi, because their DTUs have significant biological differences from one another. However, there is currently no methodological strategy for the direct typing of biological materials that has sufficient sensitivity, specificity, and reproducibility. The high diversity and copy number of the minicircle hypervariable regions (mHVRs) makes it a viable target for typing. METHODOLOGY/PRINCIPAL FINDINGS Approximately 24 million reads obtained by amplicon sequencing of the mHVR were analyzed for 62 strains belonging to the six main T. cruzi DTUs. To build reference databases of mHVR diversity for each DTU and to evaluate this target as a typing tool. Strains of the same DTU shared more mHVR clusters than strains of different DTUs, and clustered together. Different identity thresholds were used to build the reference sets of the mHVR sequences (85% and 95%, respectively). The 95% set had a higher specificity and was more suited for detecting co-infections, whereas the 85% set was excellent for identifying the primary DTU of a sample. The workflow's capacity for typing samples obtained from cultures, a set of whole-genome data, under various simulated PCR settings, in the presence of co-infecting lineages and for blood samples was also assessed. CONCLUSIONS/SIGNIFICANCE We present reference databases of mHVR sequences and an optimized typing workflow for T. cruzi including a simple online tool for deep amplicon sequencing analysis (https://ntomasini.github.io/cruzityping/). The results show that the workflow displays an equivalent resolution to that of the other typing methods. Owing to its specificity, sensitivity, relatively low cost, and simplicity, the proposed workflow could be an alternative for screening different types of samples.

中文翻译：

---

基于小环高变区的扩增子测序，用于对克氏锥虫进行分型的广泛参考数据库。

背景克氏锥虫是恰加斯病的病原体，表现出显着的遗传多样性，并被分类为不同的离散分型单位（DTU）。菌株分型技术对于研究克氏锥虫至关重要，因为它们的 DTU 彼此之间存在显着的生物学差异。然而，目前还没有具有足够敏感性、特异性和重现性的生物材料直接分型方法学策略。小环高变区 (mHVR) 的高度多样性和拷贝数使其成为可行的分型目标。方法学/主要发现 通过 mHVR 扩增子测序获得的约 2400 万条读数对属于 6 个主要克氏锥虫 DTU 的 62 个菌株进行了分析。为每个 DTU 构建 mHVR 多样性参考数据库，并评估该目标作为打字工具的效果。相同 DTU 的菌株比不同 DTU 的菌株共享更多的 mHVR 簇，并且聚集在一起。使用不同的同一性阈值来构建 mHVR 序列的参考集（分别为 85% 和 95%）。95% 组具有更高的特异性，更适合检测合并感染，而 85% 组非常适合识别样本的主要 DTU。还评估了工作流程对从培养物（一组全基因组数据）获得的样本进行分型的能力，在各种模拟 PCR 设置下，在存在共感染谱系的情况下，以及对血液样本进行分型的能力。结论/意义我们提出了 mHVR 序列的参考数据库和 T. cruzi 的优化分型工作流程，包括用于深度扩增子测序分析的简单在线工具 (https://ntomasini.github.io/cruzityping/)。结果表明，工作流程显示的分辨率与其他键入方法的分辨率相当。由于其特异性、敏感性、相对较低的成本和简单性，所提出的工作流程可以成为筛选不同类型样品的替代方案。