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Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments

Kevin P McCormick1, Matthew R Willmann2 and Blake C Meyers1*

Author Affiliations

1 Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA

2 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA

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Silence 2011, 2:2  doi:10.1186/1758-907X-2-2

Published: 28 February 2011


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.