Abstract
Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands).
Highlights
Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes
We show that smMIPs can be applied to complementary DNA (cDNA) to provide accurate estimates of differential expression, and that they are suitable for quantification of relative gene expression and allele-specific gene expression
We developed an experimental approach and dedicated statistical model (Fig. 1a) to quantify differential expression, relative expression and allelic ratios with molecule counts from cDNA-smMIPs
Summary
Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). Single-molecule molecular inversion probes (smMIPs)[8] may fill the gap between qPCR and RNA-Seq as it allows a library-free enrichment (that is, enrichment and library preparation in a single step), a high degree of multiplexing of both targets and samples, single-molecule counting via degenerate tags and the protocol to generate the sequencing library can be performed in a lab with standard PCR equipment. We show that cDNA-smMIPs are cost effective compared with alternative approaches
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