Amplicon sequencing reads for WISH-tag barcoded bacterial strains

Microbiomes feature recurrent compositional structures under given environmental conditions. However, these patterns may conceal diverse underlying population dynamics that are not captured when recording strain abundances using conventional amplicon sequencing and require intra-strain resolution. To approach this, we have chosen host microbiomes as an example and developed a novel genomic tagging system, termed WISH-tags that allows enumeration by both quantitative PCR and next-generation sequencing. We experimentally validated the performance of 62 tags and showed that they enable their differentiation with a dynamic range of at least five orders of magnitude with high precision and without background DNA amplification. We introduced WISH-tags into a set of model and non-model bacteria of the mouse and plant microbiota, respectively, and confirmed equal fitness of barcoded strains compared to untagged wild type strains. We then investigated intra-strain priority effects using isogenic barcoded bacteria in the murine gut and the Arabidopsis phyllosphere, both with and without microbiota context. Our findings reveal a resistance to colonization by late arriving strains of Salmonella Typhimurium in the mouse gut, whereas the phyllosphere accommodated latecomers of Sphingomonas in a proportional manner to their presence at the late inoculation timepoint. This illustrates how WISH-tags can serve as a valuable resource for deciphering the population dynamics underlying microbiome assembly, including bottlenecks, and can be applied across biological systems

微生物组在特定环境条件下呈现出可重复的群落组成结构。然而，这些模式可能隐藏着多样的底层种群动态——这类动态无法通过传统扩增子测序（amplicon sequencing）记录菌株丰度时被捕捉，而需要菌株内分辨率（intra-strain resolution）才能实现有效解析。为此，我们以宿主微生物组为研究范例，开发了一种新型基因组标记系统，命名为WISH-tags，该系统可通过定量PCR（quantitative PCR）与下一代测序（next-generation sequencing）实现菌株的定量计数。我们对62个标记位点的性能开展了实验验证，结果证实，这些标记可实现菌株的精准区分，动态范围至少达五个数量级，且精度极高，无背景DNA扩增现象。我们分别将WISH-tags导入小鼠和植物菌群的一组模式与非模式细菌中，并验证了带条形码的菌株与未标记的野生型菌株具有同等的生存适合度。随后，我们在有/无原生菌群背景的小鼠肠道与拟南芥叶围生境中，利用同基因条形码细菌探究了菌株内优先定植效应（intra-strain priority effects）。研究结果显示，小鼠肠道内可抵抗迟入型鼠伤寒沙门氏菌（Salmonella Typhimurium）菌株的定植，而拟南芥叶围则以与后期接种比例相符的方式容纳鞘氨醇单胞菌（Sphingomonas）的迟入菌株。这表明WISH-tags可作为一项宝贵的研究资源，用于解析微生物组组装背后的种群动态（包括定植瓶颈），且可跨多种生物系统推广应用。