Data from: Unlocking the story in the swab: a new genotyping assay for the amphibian chytrid fungus Batrachochytrium dendrobatidis

One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures has advanced our understanding of Bd phylogenetics, genomic architecture, and mechanisms of virulence. However pure cultures are laborious to obtain and whole genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus we still know little about the genetic diversity of Bd in natural systems. The most common non-invasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully-selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field-collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, non-invasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations.

野生动物中破坏性最强的新兴病原体之一为蛙壶菌（*Batrachochytrium dendrobatidis*，简称Bd），该菌已在全球范围内影响数百种两栖动物物种。基于纯培养Bd菌株获得的基因组数据，极大推动了学界对Bd系统发育、基因组结构及致病机制的认知。然而纯培养菌株的获取难度极大，且全基因组测序成本相对高昂，因此完成遗传特征鉴定的分离株数量相对有限，学界对自然生态系统中Bd的遗传多样性仍知之甚少。从自然种群中采集Bd样本最常用的非侵入式方法为擦拭两栖动物皮肤。全球范围内已从两栖动物身上采集了数十万份皮肤拭子，但通过拭子获取的Bd DNA往往存在质量和/或浓度偏低的问题。本研究借助Fluidigm Access Array平台开发了定制化Bd基因分型检测方法，用于扩增Bd基因组中192个经过精心筛选的区域，并成功为纯培养Bd菌株与野外采集的皮肤拭子样本获得了可靠的测序数据。该新型检测方法可精准区分主要的Bd进化枝，重现了此前通过全基因组数据揭示的基础系统发育树拓扑结构。此外，本研究为拭子样本中的初始Bd载量设定了临界值（150个Bd基因组当量），当样本载量高于该临界值时，本检测方法可稳定发挥作用。借助微流控多重PCR技术的进步与全球分布的两栖动物皮肤拭子样本库，非侵入式皮肤拭子样本如今可用于解答有关Bd及其对种群数量衰退两栖动物影响的关键空间与时间维度研究问题。