Strains used in this work.

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.

背景
克氏锥虫（Trypanosoma cruzi）是恰加斯病（Chagas Disease）的病原体，其遗传多样性极高，可被划分为不同的离散分型单元（Discrete Typing Units, DTUs）。虫株分型技术对克氏锥虫的研究至关重要，因为不同DTU的克氏锥虫存在显著的生物学差异。但目前尚无针对生物材料的直接分型方法，能够同时满足足够的灵敏度、特异性与可重复性。锥虫动基体微环高变区（minicircle hypervariable regions, mHVRs）的高多样性与拷贝数特性，使其成为分型研究的理想靶点。

研究方法与主要结果
本研究针对隶属于6种主要克氏锥虫DTU的62株虫株，对其mHVR扩增子测序得到的约2400万条reads进行了分析，旨在构建针对各DTU的mHVR多样性参考数据库，并评估该靶点作为分型工具的可行性。结果显示，同一DTU的虫株相较于不同DTU的虫株，共享更多的mHVR聚类群，且会聚集为独立类群。本研究分别以85%和95%的序列同一性阈值构建了mHVR序列参考集：95%同一性阈值的参考集特异性更高，更适用于检测共感染情况；而85%同一性阈值的参考集则可精准识别样本中的主要DTU。此外，本研究还评估了该分型流程在多种场景下的适用性，包括培养样本、全基因组数据集、不同模拟PCR条件、存在共感染谱系的样本以及血液样本的分型。

结论与意义
本研究构建了克氏锥虫mHVR序列参考数据库，并开发了一套优化的克氏锥虫分型流程，其中包含一款用于深度扩增子测序分析的简易在线工具（https://ntomasini.github.io/cruzityping/）。研究结果表明，该分型流程的分辨能力与其他现有分型方法相当。凭借其特异性、灵敏度、相对较低的成本与操作简便性，本研究提出的分型流程可作为不同类型样本筛查的替代方案。