Abstract
The numbers of reads generated by second-generation sequencing technologies permit to establish in a single sequencing lane multiple microRNA (miRNA) expression profiles from small RNA-derived cDNA libraries tagged by barcodes consisting of few bases. Multiplex sequencing allows sample size expansion and thus the statistical reliability of generated data. This allows the detection of discrete changes in miRNA expression levels that occur at the onset of cellular processes. With the development of the “by-amplification” strategy, tagging cDNA libraries is no more a source of technical variability. However, other specific features should be kept in mind when designing experiments aimed at profiling miRNA expression using Illumina sequencing technology, the most important being the substantial distortion between miRNA expression in sequencing data and the true miRNA abundancy. miRNAs of low expression in profiles may correspond to abundant miRNAs in samples and vice versa. We report here data obtained from rat cerebellum and liver that illustrate 1) the high 3’ adaptor dependency of miRNA expression profiles, 2) the impact of sample size when working with moderate (3 - 4 fold) changes of miRNA expression and 3) the impact of the statistical tools used to identify differentially expressed miRNAs.
Highlights
In the last decade, three next-generation sequencing (NGS) technologies have been released revolutionizing the fieldHow to cite this paper: Baroin-Tourancheau, A., Benigni, X., Doubi-Kadmiri, S., Taouis, M. and Amar, L. (2016) Lessons from microRNA Sequencing Using Illumina Technology
The relatively limited number of miRNAs, allied to the high sequencing capacity of Illumina technology, allows multiplex sequencing to be performed increasing the number of individual miRNA profiles
We developed a “by-amplification” multiplexing strategy that uses bulged primers (BP) strategy with a two-base insertion within the 3’ adaptor (see Figure 1(b))
Summary
Three next-generation sequencing (NGS) technologies (the Roche/454 GS FLX, llumina/ Solexa Genome Analyzer and Applied Biosystems SOLiD system) have been released revolutionizing the fieldHow to cite this paper: Baroin-Tourancheau, A., Benigni, X., Doubi-Kadmiri, S., Taouis, M. and Amar, L. (2016) Lessons from microRNA Sequencing Using Illumina Technology. Illumina sequencing technology that currently generates hundreds millions of reads of 40 - 100 bases per flow cell lane is well adapted to investigate small RNA transcriptomes. MicroRNAs (miRNAs) are 18 - 24 bases non-coding RNAs that act as key regulators of gene expression in eukaryotes [4]. The relatively limited number of miRNAs, allied to the high sequencing capacity of Illumina technology, allows multiplex sequencing to be performed increasing the number of individual miRNA profiles. This is a great advantage given the fact that profiling of individual miRNA transcriptomes is a major issue to recover complete genetic and epigenetic information. Multiplex sequencing gives access to any miRNA transcriptome at a relatively low cost
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