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A molecular inversion probe assay for detecting alternative splicing.

ABSTRACT: A sensitive, high-throughput method for monitoring pre-mRNA splicing on a genomic scale is needed to understand the spectrum of alternatively spliced mRNA in human cells.We adapted Molecular Inversion Probes (MIPs), a padlock-probe based technology, for the multiplexed capture and quantitation of individual splice events in human tissues. Individual MIP capture probes can be quantified using either DNA microarrays or high-throughput sequencing, which permits independent assessment of each spliced junction. Using our methodology we successfully identified 100% of our positive controls and showed that there is a strong correlation between the data from our alternative splicing MIP (asMIP) assay and quantitative PCR.The asMIP assay provides a sensitive, accurate and multiplexed means for measuring pre-mRNA splicing. Fully optimized, we estimate that the assay could accommodate a throughput of greater than 20,000 splice junctions in a single reaction. This would represent a significant improvement over existing technologies.

SUBMITTER: Lin S

PROVIDER: S-EPMC3022918 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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A molecular inversion probe assay for detecting alternative splicing.

Lin Shengrong S Wang Wenyi W Palm Curtis C Davis Ronald W RW Juneau Kara K

BMC genomics 20101217

<h4>Background</h4>A sensitive, high-throughput method for monitoring pre-mRNA splicing on a genomic scale is needed to understand the spectrum of alternatively spliced mRNA in human cells.<h4>Results</h4>We adapted Molecular Inversion Probes (MIPs), a padlock-probe based technology, for the multiplexed capture and quantitation of individual splice events in human tissues. Individual MIP capture probes can be quantified using either DNA microarrays or high-throughput sequencing, which permits in ...[more]

PMID: 21167051

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Project description:The emergence of single-cell RNA-seq (scRNA-seq) technology has made it possible to measure gene expression variations at cellular level. This breakthrough enables the investigation of a wider range of problems including analysis of splicing heterogeneity among individual cells. However, compared to bulk RNA-seq, scRNA-seq data are much noisier due to high technical variability and low sequencing depth. Here we propose SCATS (Single-Cell Analysis of Transcript Splicing) for differential splicing analysis in scRNA-seq, which achieves high sensitivity at low coverage by accounting for technical noise. SCATS models scRNA-seq data either with or without Unique Molecular Identifiers (UMIs). For non-UMI data, SCATS explicitly models technical noise by accounting for capture efficiency and amplification bias through the use of external spike-ins; for UMI data, SCATS models capture efficiency and further accounts for transcriptional burstiness. A key aspect of SCATS lies in its ability to group "exons" that originate from the same isoform(s). Grouping exons is essential in splicing analysis of scRNA-seq data as it naturally aggregates spliced reads across different exons, making it possible to detect splicing events even when sequencing depth is low. To evaluate the performance of SCATS, we analyzed both simulated and real scRNA-seq datasets and compared with existing methods including Census and DEXSeq. We show that SCATS has well controlled type I error rate, and is more powerful than existing methods, especially when splicing difference is small. In contrast, Census suffers from severe type I error inflation, whereas DEXSeq is more conservative. When applied to mouse brain scRNA-seq datasets, SCATS identified more differential splicing events with subtle difference across cell types compared to Census and DEXSeq. With the increasing adoption of scRNA-seq, we believe SCATS will be well-suited for various splicing studies. The implementation of SCATS can be downloaded from https://github.com/huyustats/SCATS.

Dataset Information

A molecular inversion probe assay for detecting alternative splicing.

Publications

A molecular inversion probe assay for detecting alternative splicing.

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