Doing Georgia, Shanbhag Priya, Bell Isaac, Cassidy Sara, Motakis Efthymios, Aiken Elizabeth, Oh Julia, Adams Mark D
The Jackson Laboratory for Genomic Medicine Farmington, Farmington, Connecticut, USA.
mSphere. 2025 May 27;10(5):e0098424. doi: 10.1128/msphere.00984-24. Epub 2025 Apr 22.
Metagenome sequencing enables the genetic characterization of complex microbial communities. However, determining the activity of isolates within a community presents several challenges, including the wide range of organismal and gene expression abundances, the presence of host RNA, and low microbial biomass at many sites. To address these limitations, we developed "targeted expression analysis sequencing" or TEAL-seq, enabling sensitive species-specific analyses of gene expression using highly multiplexed custom probe pools. For proof of concept, we targeted about 1,700 core and accessory genes of and , two key species of the skin microbiome. Two targeting methods were applied to laboratory cultures and human nasal swab specimens. Both methods showed a high degree of specificity, with >90% reads on target, even in the presence of complex microbial or human background DNA/RNA. Targeting using molecular inversion probes demonstrated excellent correlation in inferred expression levels with bulk RNA-seq. Furthermore, we show that a linear pre-amplification step to increase the number of nucleic acids for analysis yielded consistent and predictable results when applied to complex samples and enabled profiling of expression from as little as 1 ng of total RNA. TEAL-seq is much less expensive than bulk metatranscriptomic profiling, enables detection across a greater dynamic range, and uses a strategy that is readily configurable for determining the transcriptional status of organisms in any microbial community.IMPORTANCEThe gene expression patterns of bacteria in microbial communities reflect their activity and interactions with other community members. Measuring gene expression in complex microbiome contexts is challenging, however, due to the large dynamic range of microbial abundances and transcript levels. Here we describe an approach to assessing gene expression for specific species of interest using highly multiplexed pools of targeting probes. We show that an isothermal amplification step enables the profiling of low biomass samples. TEAL-seq should be widely adaptable to the study of microbial activity in natural environments.
宏基因组测序能够对复杂的微生物群落进行基因特征分析。然而，确定群落中分离菌株的活性存在诸多挑战，包括生物和基因表达丰度范围广泛、宿主RNA的存在以及许多位点的微生物生物量较低。为了解决这些限制，我们开发了“靶向表达分析测序”或TEAL-seq，它能够使用高度多重的定制探针池对基因表达进行敏感的物种特异性分析。为了验证概念，我们靶向了皮肤微生物群的两个关键物种——痤疮丙酸杆菌和金黄色葡萄球菌的约1700个核心基因和辅助基因。两种靶向方法应用于实验室培养物和人类鼻拭子标本。两种方法都显示出高度的特异性，即使在存在复杂的微生物或人类背景DNA/RNA的情况下，目标上的读数也超过90%。使用分子倒置探针进行靶向在推断的表达水平与大量RNA测序之间显示出极好的相关性。此外，我们表明，当应用于复杂样品时，增加用于分析的核酸数量的线性预扩增步骤产生了一致且可预测的结果，并能够从低至1 ng总RNA中进行表达谱分析。TEAL-seq比大量的宏转录组分析便宜得多，能够在更大的动态范围内进行检测，并且使用一种易于配置的策略来确定任何微生物群落中生物体的转录状态。
微生物群落中细菌的基因表达模式反映了它们的活性以及与其他群落成员的相互作用。然而，由于微生物丰度和转录水平的动态范围很大，在复杂的微生物组环境中测量基因表达具有挑战性。在这里，我们描述了一种使用高度多重的靶向探针池来评估特定感兴趣物种基因表达的方法。我们表明，等温扩增步骤能够对低生物量样品进行表达谱分析。TEAL-seq应该广泛适用于自然环境中微生物活性的研究。
