Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing - PubMed (original) (raw)

Comparative Study

. 2009 Nov;15(11):2028-34.

doi: 10.1261/rna.1699809. Epub 2009 Sep 10.

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Comparative Study

Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing

Hanni Willenbrock et al. RNA. 2009 Nov.

Abstract

Recently, next-generation sequencing has been introduced as a promising, new platform for assessing the copy number of transcripts, while the existing microarray technology is considered less reliable for absolute, quantitative expression measurements. Nonetheless, so far, results from the two technologies have only been compared based on biological data, leading to the conclusion that, although they are somewhat correlated, expression values differ significantly. Here, we use synthetic RNA samples, resembling human microRNA samples, to find that microarray expression measures actually correlate better with sample RNA content than expression measures obtained from sequencing data. In addition, microarrays appear highly sensitive and perform equivalently to next-generation sequencing in terms of reproducibility and relative ratio quantification.

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Figures

FIGURE 1.

FIGURE 1.

Comparison of microarray and sequencing data. (A,B) Ninety-five percent confidence intervals for sample A intensities versus RNA concentrations. (C) Illumina sequencing ratios versus microarray ratios. (D) Bar plot of the undetected fraction (false discovery rate) of synthetic RNAs at each concentration. The lower the bar, the better sensitivity.

FIGURE 2.

FIGURE 2.

Histogram of read variants’ lengths in Illumina sequencing data. The black bar shows the number of exact sequence read matches for all synthetic RNAs, while the gray bars show the number of length variants that are perfect matches but shorter or longer than the synthetic RNA it resembles the most. “Relative length” is the read length relative to the length of the synthetic RNA it resembles the most.

FIGURE 3.

FIGURE 3.

Histogram of the alignment distances (sum of mismatches and gaps in alignments) between the human let-7 family sequences (black) according to the miRBase sequences and unique variant reads aligning to the let-7 family members (gray) as its best match. In comparison, many variant read sequences have much higher alignment distances to their closest matching synthetic RNA sequence than alignment distances observed within the let-7 miRNA family.

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