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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Bibliographic Details
Published in:Silence 2011-02, Vol.2 (1), p.2-2, Article 2
Main Authors: McCormick, Kevin P, Willmann, Matthew R, Meyers, Blake C
Format: Article
Language:English
Subjects:
DNA sequencing
Gene expression
Nucleotide sequencing
Physiological aspects
Review
RNA
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Summary: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.
ISSN:1758-907X
1758-907X
DOI:10.1186/1758-907x-2-2