RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries
- Markus Hafner1,
- Neil Renwick1,
- Miguel Brown1,
- Aleksandra Mihailović1,
- Daniel Holoch1,
- Carolina Lin1,
- John T.G. Pena1,2,
- Jeffrey D. Nusbaum1,
- Pavel Morozov1,
- Janos Ludwig1,
- Tolulope Ojo1,
- Shujun Luo3,
- Gary Schroth3 and
- Thomas Tuschl1,4
- 1Howard Hughes Medical Institute, Laboratory for RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA
- 2Weill Cornell Medical College, Dyson Vision Research Institute, New York, New York 10065, USA
- 3Illumina, Inc., Hayward, California 94545, USA
Abstract
Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3′ and 5′ ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1–249) and Rnl2(1–249)K227Q, for 3′-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5′-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3′-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5′-terminal 5-nt barcode extensions for a set of 20 barcoded 3′ adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.
Keywords
- small RNAs
- high-throughput sequencing
- next-generation sequencing
- sequence-specific bias
- adapter ligation
- oligonucleotide linker ligation
Footnotes
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↵4 Corresponding author.
E-mail ttuschl{at}rockefeller.edu.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2799511.
- Received May 4, 2011.
- Accepted June 10, 2011.
- Copyright © 2011 RNA Society