Design and application of a novel two-amplicon approach for defining eukaryotic microbiota

Microbiome

David S. Guttman¹  Pauline W. Wang¹  Michael E. Grigg²  John Parkinson³  Ana Popovic³  Robert H. J. Bandsma⁴  Celine Bourdon⁴  Wieger Voskuijl⁵ 

[1]Department of Cell and Systems Biology, University of Toronto

[2]Molecular Parasitology Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health

[3]Program in Molecular Medicine, The Hospital for Sick Children, Peter Gilgan Centre for Research and Learning

[4]Program in Translational Medicine, The Hospital for Sick Children, Peter Gilgan Center for Research and Learning

[5]The Childhood Acute Illness & Nutrition Network (CHAIN), Nairobi Coordination Centre

关键词: Microbiome;    Eukaryotes;    Parasites;    Next-generation sequencing;    Amplicon sequencing;    Malnutrition;   

DOI  :  10.1186/s40168-018-0612-3

来源: DOAJ

【 摘 要 】

Abstract Background Due to a lack of systematic diagnostics, our understanding of the diversity and role of eukaryotic microbiota in human health is limited. While studies have shown fungal communities to be significant modulators of human health, information on the prevalence of taxa such as protozoa and helminths has been limited to a small number of species for which targeted molecular diagnostics are available. To probe the diversity of eukaryotic microbes and their relationships with other members of the microbiota, we applied in silico and experimental approaches to design a novel two-amplicon surveillance tool, based on sequencing regions of ribosomal RNA genes and their internal transcribed spacers. We subsequently demonstrated the utility of our approach by characterizing the eukaryotic microbiota of 46 hospitalized Malawian children suffering from Severe Acute Malnutrition (SAM). Results Through in silico analysis and validation on a diverse panel of eukaryotes, we identified 18S rRNA variable genetic regions 4 and 5 (18S V4 V5), together with a region encoding 28S rRNA variable genetic region 2 and the internal transcribed spacers (transITS), as optimal for the systematic classification of eukaryotes. Sequencing of these regions revealed protozoa in all stool samples from children with SAM and helminths in most, including several eukaryotes previously implicated in malnutrition and diarrheal disease. Clinical comparisons revealed no association between protozoan parasites and diarrhea or HIV reactivity. However, the presence of Blastocystis correlated with bacterial alpha diversity and increased abundance of specific taxa, including Sporobacter, Cellulosibacter, Oscillibacter, and Roseburia. Conclusion We suggest this novel two-amplicon based strategy will prove an effective tool to deliver new insights into the role of eukaryotic microbiota in health and disease.

【 授权许可】

Unknown