Abstract
BackgroundHigh-throughput sequencing is gradually replacing microarrays as the preferred method for studying mRNA expression levels, providing nucleotide resolution and accurately measuring absolute expression levels of almost any transcript, known or novel. However, existing microarray data from clinical, pharmaceutical, and academic settings represent valuable and often underappreciated resources, and methods for assessing and improving the quality of these data are lacking.ResultsTo quantitatively assess the quality of microarray probes, we directly compare RNA-Seq to Agilent microarrays by processing 231 unique samples from the Allen Human Brain Atlas using RNA-Seq. Both techniques provide highly consistent, highly reproducible gene expression measurements in adult human brain, with RNA-Seq slightly outperforming microarray results overall. We show that RNA-Seq can be used as ground truth to assess the reliability of most microarray probes, remove probes with off-target effects, and scale probe intensities to match the expression levels identified by RNA-Seq. These sequencing scaled microarray intensities (SSMIs) provide more reliable, quantitative estimates of absolute expression levels for many genes when compared with unscaled intensities. Finally, we validate this result in two human cell lines, showing that linear scaling factors can be applied across experiments using the same microarray platform.ConclusionsMicroarrays provide consistent, reproducible gene expression measurements, which are improved using RNA-Seq as ground truth. We expect that our strategy could be used to improve probe quality for many data sets from major existing repositories.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-154) contains supplementary material, which is available to authorized users.
Highlights
High-throughput sequencing is gradually replacing microarrays as the preferred method for studying mRNA expression levels, providing nucleotide resolution and accurately measuring absolute expression levels of almost any transcript, known or novel
To directly compare the output of transcriptome analysis from microarrays and RNA-Seq across the human brain, we reanalyzed a subset of the same RNA isolates used for microarray analysis using RNA-Seq
In total each brain region was analyzed in eight independent samples, spanning both hemispheres of both brains, with two independent sampling sites per hemisphere
Summary
High-throughput sequencing is gradually replacing microarrays as the preferred method for studying mRNA expression levels, providing nucleotide resolution and accurately measuring absolute expression levels of almost any transcript, known or novel. RNA-Seq and related sequencing-based technologies are gradually emerging as the preferred method for genomewide transcriptional analyses, as they provide several potential advantages over hybridization-based microarray technologies [1,2,3,4,5]. Fragment counts from RNA-Seq more reliably track absolute gene expression levels (as measured by quantitative PCR) than the fluorescence- or intensity-based measures obtained using DNA microarrays [2,3]. As RNA-Seq does not require a priori probe selection, it allows unbiased analysis of the entire transcriptome, including measurements of gene isoforms, noncoding RNAs, novel transcripts [4], and base-level transcriptional changes. Data from thousands of studies on all of the major microarray platforms are publicly available in databases such as ArrayExpress [8] and
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