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Reducing ligation bias of small RNAs in libraries for next generation sequencing
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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489589/ https://www.ncbi.nlm.nih.gov/pubmed/22647250 http://dx.doi.org/10.1186/1758-907X-3-4 |
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author | Sorefan, Karim Pais, Helio Hall, Adam E Kozomara, Ana Griffiths-Jones, Sam Moulton, Vincent Dalmay, Tamas |
author_facet | Sorefan, Karim Pais, Helio Hall, Adam E Kozomara, Ana Griffiths-Jones, Sam Moulton, Vincent Dalmay, Tamas |
author_sort | Sorefan, Karim |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3489589 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-34895892012-11-06 Reducing ligation bias of small RNAs in libraries for next generation sequencing Sorefan, Karim Pais, Helio Hall, Adam E Kozomara, Ana Griffiths-Jones, Sam Moulton, Vincent Dalmay, Tamas Silence Short Report 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. BioMed Central 2012-05-30 /pmc/articles/PMC3489589/ /pubmed/22647250 http://dx.doi.org/10.1186/1758-907X-3-4 Text en Copyright ©2012 Sorefan et al.; licensee BioMed Central Ltd http://creativecommons.org/licenses/by/2.0 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. |
spellingShingle | Short Report Sorefan, Karim Pais, Helio Hall, Adam E Kozomara, Ana Griffiths-Jones, Sam Moulton, Vincent Dalmay, Tamas Reducing ligation bias of small RNAs in libraries for next generation sequencing |
title | Reducing ligation bias of small RNAs in libraries for next generation sequencing |
title_full | Reducing ligation bias of small RNAs in libraries for next generation sequencing |
title_fullStr | Reducing ligation bias of small RNAs in libraries for next generation sequencing |
title_full_unstemmed | Reducing ligation bias of small RNAs in libraries for next generation sequencing |
title_short | Reducing ligation bias of small RNAs in libraries for next generation sequencing |
title_sort | reducing ligation bias of small rnas in libraries for next generation sequencing |
topic | Short Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489589/ https://www.ncbi.nlm.nih.gov/pubmed/22647250 http://dx.doi.org/10.1186/1758-907X-3-4 |
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