Barcoding bias in high-throughput multiplex sequencing of miRNA
- Shahar Alon1,6,
- Francois Vigneault2,3,4,6,
- Seda Eminaga2,
- Danos C. Christodoulou2,
- Jonathan G. Seidman2,
- George M. Church2,3 and
- Eli Eisenberg5,7
- 1Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel;
- 2Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA;
- 3Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts 02115, USA;
- 4Ragon Institute of MGH, MIT, and Harvard, Boston, Massachusetts 02129, USA;
- 5Raymond and Beverly Sackler School of Physics and Astronomy, Tel-Aviv University, Tel-Aviv 69978, Israel
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↵6 These authors contributed equally to this work.
Abstract
Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number.
Footnotes
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↵7 Corresponding author.
E-mail elieis{at}post.tau.ac.il.
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.121715.111.
- Received February 3, 2011.
- Accepted July 5, 2011.
- Copyright © 2011 by Cold Spring Harbor Laboratory Press
