Abstract
Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559–0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.
Highlights
Dual species transcriptomic experiments have been increasingly employed as a method to analyze the transcriptomes of multiple species within a system[1,2,3,4,5,6,7,8,9]
To test the efficacy of the Agilent SureSelect (AgSS) platform on dual- and tripartite-species systems where insufficient numbers of reads are obtained from the minor member(s), we designed AgSS baits for: (1) Brugia malayi, a filarial nematode and the causative agent of lymphatic filariasis, (2) wBm, the obligate mutualistic Wolbachia endosymbiont of B. malayi, and (3) Aspergillus fumigatus AF293, a fungal pathogen known to cause aspergillosis in immunocompromised individuals
We explored whether sequencing data from both the major and minor organism could be accurately obtained using only data collected from a AgSS capture of the minor organism for eukaryote-eukaryote dual-transcriptomics experiments
Summary
Dual species transcriptomic experiments have been increasingly employed as a method to analyze the transcriptomes of multiple species within a system[1,2,3,4,5,6,7,8,9]. Whole infected mosquito thoraces containing the larval B. malayi are used for RNA isolation As such the B. malayi reads are overwhelmed by reads from those of the mosquito vector, typically experimentally infected Aedes aegypti, and the reads of the endogenous wBm are even further dwarfed compared to samples obtained from the definitive host. In a separate dual-species transcriptomics experiment examining A fumigatus infections, the eukaryotic A. fumigatus reads are overwhelmed by reads from the eukaryotic human or mouse host making it difficult to analyze the fungal transcriptome in this host-pathogen interaction Given that both are eukaryotes there are not differences in characteristics like polyadenylation to enable the enrichment of the mRNA from the minor member, A. fumigatus. The efficacy of the AgSS platform in extracting prokaryotic reads was determined by comparing the total RNA AgSS capture to the RiboZero-treated, poly(A)-depletion method in extracting wBm reads
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