The electronic tree of life (eToL): a net of long probes to characterize the microbiome from RNA-seq data

BMC Microbiology

Research

Richard Lathe¹  Jürgen G. Haas¹  Xinyue Hu² 

[1] Division of Infection Medicine, University of Edinburgh, Little France, EH16 4SB, Edinburgh, UK;Program in Bioinformatics, School of Biological Sciences, King’s Buildings, University of Edinburgh, EH9 3FD, Edinburgh, UK;

关键词: Archaea;    Bacteria;    BLAST;    brain;    disease;    Fungi;    microbiome;    RNA-seq;    Tree of Life;    virus;   

DOI  :  10.1186/s12866-022-02671-2

 received in 2021-12-27, accepted in 2022-10-11,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundMicrobiome analysis generally requires PCR-based or metagenomic shotgun sequencing, sophisticated programs, and large volumes of data. Alternative approaches based on widely available RNA-seq data are constrained because of sequence similarities between the transcriptomes of microbes/viruses and those of the host, compounded by the extreme abundance of host sequences in such libraries. Current approaches are also limited to specific microbial groups. There is a need for alternative methods of microbiome analysis that encompass the entire tree of life.ResultsWe report a method to specifically retrieve non-human sequences in human tissue RNA-seq data. For cellular microbes we used a bioinformatic 'net', based on filtered 64-mer sequences designed from small subunit ribosomal RNA (rRNA) sequences across the Tree of Life (the 'electronic tree of life', eToL), to comprehensively (98%) entrap all non-human rRNA sequences present in the target tissue. Using brain as a model, retrieval of matching reads, re-exclusion of human-related sequences, followed by contig building and species identification, is followed by confirmation of the abundance and identity of the corresponding species groups. We provide methods to automate this analysis. The method reduces the computation time versus metagenomics by a factor of >1000. A variant approach is necessary for viruses. Again, because of significant matches between viral and human sequences, a 'stripping' approach is essential. Contamination during workup is a potential problem, and we discuss strategies to circumvent this issue. To illustrate the versatility of the method we report the use of the eToL methodology to unambiguously identify exogenous microbial and viral sequences in human tissue RNA-seq data across the entire tree of life including Archaea, Bacteria, Chloroplastida, basal Eukaryota, Fungi, and Holozoa/Metazoa, and discuss the technical and bioinformatic challenges involved.ConclusionsThis generic methodology is likely to find wide application in microbiome analysis including diagnostics.

【 授权许可】

CC BY   
© The Author(s) 2022

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