TEAL-Seq:靶向表达分析测序。

IF 3.7 2区 生物学 Q2 MICROBIOLOGY
mSphere Pub Date : 2025-05-27 Epub Date: 2025-04-22 DOI:10.1128/msphere.00984-24
Georgia Doing, Priya Shanbhag, Isaac Bell, Sara Cassidy, Efthymios Motakis, Elizabeth Aiken, Julia Oh, Mark D Adams
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引用次数: 0

摘要

宏基因组测序使复杂微生物群落的遗传特征。然而,确定一个群落中分离物的活性面临着几个挑战,包括广泛的生物体和基因表达丰度,宿主RNA的存在,以及许多位点的微生物生物量低。为了解决这些限制,我们开发了“靶向表达分析测序”或TEAL-seq,使用高度复用的定制探针池对基因表达进行敏感的物种特异性分析。为了验证这一概念,我们针对皮肤微生物组的两个关键物种金黄色葡萄球菌和表皮葡萄球菌的大约1,700个核心和辅助基因进行了研究。两种靶向方法分别应用于实验室培养物和人鼻拭子标本。这两种方法都显示出高度的特异性,即使在复杂的微生物或人类背景DNA/RNA存在的情况下,也能在目标上读取bbb90 %。利用分子倒置探针的靶向研究表明,推断的表达水平与大量RNA-seq具有良好的相关性。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扩增前步骤产生了一致和可预测的结果,并且可以从少至1ng的总RNA中分析表达。TEAL-seq比大量的偏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和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大动态范围,在复杂的微生物组环境中测量基因表达是具有挑战性的。在这里,我们描述了一种方法来评估基因表达的特定物种的兴趣使用高度复用池的靶向探针。我们表明,等温扩增步骤能够分析低生物量样品。TEAL-seq应该广泛适用于自然环境中微生物活动的研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
TEAL-Seq: targeted expression analysis sequencing.

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 Staphylococcus aureus and S. epidermidis, 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.

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来源期刊
mSphere
mSphere Immunology and Microbiology-Microbiology
CiteScore
8.50
自引率
2.10%
发文量
192
审稿时长
11 weeks
期刊介绍: mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.
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