title: Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: underestimated effects of contamination in NGS.
abstract: The report that exogenous plant miRNAs are able to cross the mammalian gastrointestinal tract and exert gene-regulation mechanism in mammalian tissues has yielded a lot of controversy, both in the public press and the scientific literature. Despite the initial enthusiasm, reproducibility of these results was recently questioned by several authors. To analyze the causes of this unease, we searched for diet-derived miRNAs in deep-sequencing libraries performed by ourselves and others. We found variable amounts of plant miRNAs in publicly available small RNA-seq data sets of human tissues. In human spermatozoa, exogenous RNAs reached extreme, biologically meaningless levels. On the contrary, plant miRNAs were not detected in our sequencing of human sperm cells, which was performed in the absence of any known sources of plant contamination. We designed an experiment to show that cross-contamination during library preparation is a source of exogenous RNAs. These contamination-derived exogenous sequences even resisted oxidation with sodium periodate. To test the assumption that diet-derived miRNAs were actually contamination-derived, we sought in the literature for previous sequencing reports performed by the same group which reported the initial finding. We analyzed the spectra of plant miRNAs in a small RNA sequencing study performed in amphioxus by this group in 2009 and we found a very strong correlation with the plant miRNAs which they later reported in human sera. Even though contamination with exogenous sequences may be easy to detect, cross-contamination between samples from the same organism can go completely unnoticed, possibly affecting conclusions derived from NGS transcriptomics.
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay Faculty of Science, Universidad de la República, Montevideo 11400, Uruguay.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Tosar
authors.affiliation: Division of Oncology, Department of Clinical Sciences, Lund University Cancer Center, Lund 223 81, Sweden.
authors.forename: Carlos
authors.initials: C
authors.lastname: Rovira
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay.
authors.forename: Hugo
authors.initials: H
authors.lastname: Naya
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay Faculty of Medicine, Universidad de la República, Montevideo 11800, Uruguay.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Cayota
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000378:metabolism*; D012275:Oryza / Q000235:genetics*; D018749:RNA, Plant / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D035683:MicroRNAs; D018749:RNA, Plant
keywords: contamination; diet-derived; exogenous; microRNAs; plant MIR168a
references.citation: RNA Biol. 2013 Jul;10(7):1080-6
references.pmid: 23770773
references.citation: Nucleic Acids Res. 2013 Apr;41(7):4104-17
references.pmid: 23471003
references.citation: Cell Res. 2012 Jan;22(1):107-26
references.pmid: 21931358
references.citation: Proc Natl Acad Sci U S A. 2012 Jan 10;109(2):490-4
references.pmid: 22203953
references.citation: Nat Biotechnol. 2013 Nov;31(11):967-9
references.pmid: 24213764
references.citation: Genome Biol. 2009;10(7):R78
references.pmid: 19615057
references.citation: Regul Toxicol Pharmacol. 2013 Jul;66(2):167-76
references.pmid: 23557984
references.citation: PLoS One. 2013;8(4):e60234
references.pmid: 23565205
references.citation: RNA Biol. 2013 Jul;10(7):1107-16
references.pmid: 23669076
references.citation: Nat Biotechnol. 2012 Nov;30(11):1084-94
references.pmid: 23138308
references.citation: Bioessays. 2014 Apr;36(4):394-406
references.pmid: 24436255
references.citation: N Engl J Med. 2013 Dec 19;369(25):2369-71
references.pmid: 24251383
references.citation: Nat Protoc. 2010 Mar;5(3):516-35
references.pmid: 20203668
references.citation: BMC Genomics. 2012 Aug 08;13:381
references.pmid: 22873950
references.citation: Nat Biotechnol. 2013 Nov;31(11):965-7
references.pmid: 24213763