Unit

UNIT 11.12 High-Throughput Multiplex Sequencing of miRNA

  1. Francois Vigneault1,2,3,
  2. Dmitry Ter-Ovanesyan2,4,
  3. Shahar Alon5,6,
  4. Seda Eminaga1,
  5. Danos C. Christodoulou1,
  6. J. G. Seidman1,
  7. Eli Eisenberg5,6,
  8. George M. Church1,2

Published Online: 1 APR 2012

DOI: 10.1002/0471142905.hg1112s73

Current Protocols in Human Genetics

Current Protocols in Human Genetics

How to Cite

Vigneault, F., Ter-Ovanesyan, D., Alon, S., Eminaga, S., C. Christodoulou, D., Seidman, J. G., Eisenberg, E. and M. Church, G. 2012. High-Throughput Multiplex Sequencing of miRNA. Current Protocols in Human Genetics. 73:11.12:11.12.1–11.12.10.

Author Information

  1. 1

    Department of Genetics, Harvard Medical School, Boston, Massachusetts

  2. 2

    Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts

  3. 3

    Ragon Institute of MGH, MIT, and Harvard, Charlestown, Massachusetts

  4. 4

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts

  5. 5

    Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

  6. 6

    Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel

Publication History

  1. Published Online: 1 APR 2012
  2. Published Print: APR 2012

Abstract

Next-generation sequencing offers many advantages over other methods of microRNA (miRNA) expression profiling, such as sample throughput and the capability to discover novel miRNAs. As the sequencing depth of current sequencing platforms exceeds what is necessary to quantify miRNAs, multiplexing several samples in one sequencing run offers a significant cost advantage. Although previous studies have achieved this goal by adding bar codes to miRNA libraries at the ligation step, this was recently shown to introduce significant bias into the miRNA expression data. This bias can be avoided, however, by bar coding the miRNA libraries at the PCR step instead. Here, we describe a user-friendly PCR bar-coding method of preparing multiplexed microRNA libraries for Illumina-based sequencing. The method also prevents the production of adapter dimers and can be completed in one day. Curr. Protoc. Hum. Genet. 73:11.12.1-11.12.10 © 2012 by John Wiley & Sons, Inc.

Keywords:

  • miRNA;
  • Illumina;
  • sequencing;
  • library;
  • multiplex;
  • bar code