TEAL-seq (Targeted Expression Analysis Sequencing) of S. aureus and S. epidermidis in TSB and skin-relevent conditions

Metagenome sequencing enables discovery and genetic characterization of complex microbial communities from diverse ecosystems. However, determining the activity of isolates within a community using transcriptomics presents several challenges including the wide dynamic range of organismal and gene expression abundances, the presence of host RNA, and low microbial biomass at many body sites. To address these limitations, we developed “Targeted Expression Analysis Sequencing” or TEAL-seq. Targeting strategies enabled sensitive species-specific analyses of gene expression using highly multiplexed custom probe pools targeting about 1700 core and accessory genes of Staphylococcus aureus (S.a.) and S. epidermidis (S.e.), two key species of the skin microbiome. Two targeting methods were applied to mixed cultures and nasal swab specimens from human research participants. 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. Further, we show that a linear pre-amplification step to increase the amount of input nucleic acids for analysis was quite reproducible . While pre-amplification introduced some noise compared to non-amplified samples, it also enabled profiling of expression from as little as 1 ng of total RNA. TEAL-seq is much less expensive than bulk metatranscriptomic profiling and enables detection across a greater dynamic range. Custom probe pools are readily configurable and this strategy is broadly applicable for determining the transcriptional status of organisms in any microbial community. S. aureus and S. epidermidis were grown in TSB, TSB pH 4.8 or on reconstructed human epidermidis for bulk and targted RNA-seq with and without ribo-depletion and/or SPIA.

宏基因组测序（Metagenome sequencing）能够实现对不同生态系统中复杂微生物群落的发现与遗传特征解析。然而，利用转录组学解析群落内分离株的活性面临诸多挑战，包括生物体与基因表达丰度的动态范围极广、宿主RNA的干扰，以及多数人体位点的微生物生物量较低。为解决这些局限，本研究开发了「靶向表达分析测序（Targeted Expression Analysis Sequencing，简称TEAL-seq）」。该靶向策略依托高度多重化的定制探针池，靶向金黄色葡萄球菌（Staphylococcus aureus，简称S.a.）与表皮葡萄球菌（S. epidermidis，简称S.e.）的约1700个核心及附属基因，二者均为皮肤微生物组的关键物种，可实现高灵敏度的物种特异性基因表达分析。研究将两种靶向方法应用于混合培养物以及人类研究受试者的鼻拭子样本。两种方法均展现出极高的特异性，即使存在复杂的微生物或人类背景DNA/RNA，靶标测序读段占比仍超过90%。采用分子倒置探针的靶向方法，其推断的基因表达水平与批量RNA测序（bulk RNA-seq）结果具有极佳的相关性。此外，本研究证实，为提升分析所需的输入核酸总量而采用的线性预扩增步骤具有极佳的重现性。尽管与未扩增样本相比，预扩增会引入一定噪声，但该步骤可实现仅需1 ng总RNA即可完成的表达谱分析。TEAL-seq的成本远低于批量宏转录组谱分析，且可实现更广动态范围的检测。定制探针池易于定制配置，该策略可广泛应用于解析任意微生物群落中生物体的转录状态。本研究在胰蛋白胨大豆肉汤（TSB）、pH 4.8的TSB培养基或重建人类表皮模型中培养金黄色葡萄球菌与表皮葡萄球菌，分别开展有/无核糖体去除（ribo-depletion）和/或SPIA的批量RNA测序与靶向RNA测序。