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Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments
Prior to the advent of new, deep sequencing methods, small RNA (sRNA) discovery was dependent on Sanger sequencing, which was time-consuming and limited knowledge to only the most abundant sRNA. The innovation of large-scale, next-generation sequencing has exponentially increased knowledge of the bi...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2011
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055805/ https://www.ncbi.nlm.nih.gov/pubmed/21356093 http://dx.doi.org/10.1186/1758-907X-2-2 |
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author | McCormick, Kevin P Willmann, Matthew R Meyers, Blake C |
author_facet | McCormick, Kevin P Willmann, Matthew R Meyers, Blake C |
author_sort | McCormick, Kevin P |
collection | PubMed |
description | Prior to the advent of new, deep sequencing methods, small RNA (sRNA) discovery was dependent on Sanger sequencing, which was time-consuming and limited knowledge to only the most abundant sRNA. The innovation of large-scale, next-generation sequencing has exponentially increased knowledge of the biology, diversity and abundance of sRNA populations. In this review, we discuss issues involved in the design of sRNA sequencing experiments, including choosing a sequencing platform, inherent biases that affect sRNA measurements and replication. We outline the steps involved in preprocessing sRNA sequencing data and review both the principles behind and the current options for normalization. Finally, we discuss differential expression analysis in the absence and presence of biological replicates. While our focus is on sRNA sequencing experiments, many of the principles discussed are applicable to the sequencing of other RNA populations. |
format | Text |
id | pubmed-3055805 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-30558052011-03-12 Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments McCormick, Kevin P Willmann, Matthew R Meyers, Blake C Silence Review Prior to the advent of new, deep sequencing methods, small RNA (sRNA) discovery was dependent on Sanger sequencing, which was time-consuming and limited knowledge to only the most abundant sRNA. The innovation of large-scale, next-generation sequencing has exponentially increased knowledge of the biology, diversity and abundance of sRNA populations. In this review, we discuss issues involved in the design of sRNA sequencing experiments, including choosing a sequencing platform, inherent biases that affect sRNA measurements and replication. We outline the steps involved in preprocessing sRNA sequencing data and review both the principles behind and the current options for normalization. Finally, we discuss differential expression analysis in the absence and presence of biological replicates. While our focus is on sRNA sequencing experiments, many of the principles discussed are applicable to the sequencing of other RNA populations. BioMed Central 2011-02-28 /pmc/articles/PMC3055805/ /pubmed/21356093 http://dx.doi.org/10.1186/1758-907X-2-2 Text en Copyright ©2011 McCormick 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 | Review McCormick, Kevin P Willmann, Matthew R Meyers, Blake C Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments |
title | Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments |
title_full | Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments |
title_fullStr | Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments |
title_full_unstemmed | Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments |
title_short | Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments |
title_sort | experimental design, preprocessing, normalization and differential expression analysis of small rna sequencing experiments |
topic | Review |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055805/ https://www.ncbi.nlm.nih.gov/pubmed/21356093 http://dx.doi.org/10.1186/1758-907X-2-2 |
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