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Jackson Laboratoryhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1171333xref:PubMed:40261045Metagenome 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. Overall design: 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.ENAfalseTEAL-seq (Targeted Expression Analysis Sequencing) of S. aureus and S. epidermidis in TSB and skin-relevent conditionsTEAL-seq (Targeted Expression Analysis Sequencing) of S. aureus and S. epidermidis in TSB and skin-relevent conditions2025-06-072024-10-29PRJNA1171333GSE27918740261045