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.

Keywords: contamination; diet-derived; exogenous; microRNAs; plant MIR168a.

FIGURE 1.

Relative abundance of putative exogenous sequences in sRNA data sets from human tissues (A,B), and correlation between plant miRNAs in human sera and in amphioxus (C,D). (A) Reads corresponding to plant MIR168a (green) or other exogenous sequences (red) were normalized to the total number of reads corresponding to human miRNAs in each sample and multiplied by 100. Abbreviations: LUN, lung; ORB, orbital gyrus; PAN, pancreas; SPL, spleen; KID, kidney; LIV, liver; HBV, HBV-infected liver; SCH, liver with severe chronic hepatitis B; B-M, bone marrow; CD34, CD34+ progenitor blood cells; THYM, thymocytes; SKI, skin; SPE, spermatozoa. (B) For reasons of scale, spermatozoa samples were presented separately. (C) Correlation between the levels of plant miRNAs (normalized to 1 million mammalian miRNAs) found in human serum (“male” library) according to Zhang et al. (2012a) and their corresponding levels (raw reads) found in previous data from the same group (GSE16859; small RNA sequencing in amphioxus). Pearson r = 0.9874; P < 0.0001; Spearman rho = 0.9439; P < 0.0001 (n = 11; two-tailed P-values). (D) Spearman's rank correlation coefficient (rho) values were calculated for the correlation between the 10 human samples sequenced by Zhang and colleagues and their corresponding levels in amphioxus. The red and blue lines show the critical values (n = 11) for P < 0.05 and P < 0.005, respectively.