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Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing

Nature Methods volume 11, pages 51–54 (2014)Cite this article

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Abstract

We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

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Figure 1: The development and performance of mmPCR-seq.
Figure 2: Characterization of novel RNA-editing sites identified by mmPCR-seq.
Figure 3: ASE analysis of mmPCR-seq data.

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Acknowledgements

We thank M. Snyder for access to the Fluidigm Access Array system and W. Sun for advice on TReCASE analysis. R.Z. was partially supported by a Dean's fellowship from Stanford University School of Medicine. G.R. was supported by a Stanford Graduate Fellowship. This work was supported by the US National Institutes of Health (GM102484); Ellison Medical Foundation and United States–Israel Binational Science Foundation (to J.B.L.); and Edward Mallinckrodt, Jr. Foundation (to S.B.M.).

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Authors and Affiliations

  1. Department of Genetics, Stanford University, Stanford, California, USA

    Rui Zhang, Gokul Ramaswami, Stephen B Montgomery & Jin Billy Li

  2. Department of Pathology, Stanford University, Stanford, California, USA

    Xin Li, Kevin S Smith & Stephen B Montgomery

  3. McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada

    Gustavo Turecki

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  1. Rui Zhang
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Contributions

R.Z. developed and optimized the mmPCR-seq method with the help from G.R., K.S.S., S.B.M. and J.B.L. R.Z. and X.L. performed computational analyses with help from S.B.M. and J.B.L. G.T. provided the brain samples. R.Z., X.L., S.B.M. and J.B.L. wrote the paper.

Corresponding authors

Correspondence to Stephen B Montgomery or Jin Billy Li.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17, Supplementary Tables 1–9 and Supplementary Notes 1–6 (PDF 2193 kb)

Supplementary Data 1

Targeted RNA editing sites and ASE sites (XLSX 58 kb)

Supplementary Data 2

Primer information (XLSX 81 kb)

Supplementary Data 3

Validation of A-to-I events (XLSX 51 kb)

Supplementary Data 4

Known and novel nonrepetitive recoding sites (XLSX 19 kb)

Supplementary Software

Perl script for multiplex PCR primer design (ZIP 3389 kb)

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Zhang, R., Li, X., Ramaswami, G. et al. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing. Nat Methods 11, 51–54 (2014). https://doi.org/10.1038/nmeth.2736

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