Brief Communication | Published:

Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing

Nature Methods volume 11, pages 5154 (2014) | Download Citation

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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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.).

Author information

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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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Stephen B Montgomery or Jin Billy Li.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–17, Supplementary Tables 1–9 and Supplementary Notes 1–6

Excel files

  1. 1.

    Supplementary Data 1

    Targeted RNA editing sites and ASE sites

  2. 2.

    Supplementary Data 2

    Primer information

  3. 3.

    Supplementary Data 3

    Validation of A-to-I events

  4. 4.

    Supplementary Data 4

    Known and novel nonrepetitive recoding sites

Zip files

  1. 1.

    Supplementary Software

    Perl script for multiplex PCR primer design

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DOI

https://doi.org/10.1038/nmeth.2736

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