Sorefan, K. orcid.org/0000-0002-6266-6022, Pais, H., Hall, A.E. et al. (4 more authors) (2012) Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence, 3. 4. ISSN 1758-907X
Abstract
BACKGROUND: The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing. FINDINGS: We demonstrate that sequencing data of libraries generated by RNA ligases can reveal novel secondary structure preferences of these enzymes, which are used in small RNA cloning and library preparation for NGS. Using this knowledge we demonstrate that the cloning bias in small RNA libraries is RNA ligase-dependent. We developed a high definition (HD) protocol that reduces the RNA ligase-dependent cloning bias. The HD protocol doubled read coverage, is quantitative and found previously unidentified microRNAs. In addition, we show that microRNAs in miRBase are those preferred by the adapters of the main sequencing platform. CONCLUSIONS: Sequencing bias of small RNAs partially influenced which microRNAs have been studied in depth; therefore most previous small RNA profiling experiments should be re-evaluated. New microRNAs are likely to be found, which were selected against by existing adapters. Preference of currently used adapters towards known microRNAs suggests that the annotation of all existing small RNAs, including miRNAs, siRNAs and piRNAs, has been biased.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © Sorefan et al.; licensee BioMed Central Ltd 2012 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Next generation sequencing; MicroRNA; Small RNA; MiRBase; Expression profile; Deep sequencing; T4 RNA ligase |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 14 Apr 2016 10:45 |
Last Modified: | 23 Jun 2017 15:29 |
Published Version: | http://dx.doi.org/10.1186/1758-907X-3-4 |
Status: | Published |
Publisher: | BioMed Central |
Refereed: | Yes |
Identification Number: | 10.1186/1758-907X-3-4 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:98511 |