Capturing the 'ome': the expanding molecular toolbox for RNA and DNA library construction.

Morgane Boone,Andries De Koker,Nico Callewaert

doi:10.1093/nar/gky167

Abstract

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application.

Highlights

Generation sequencing technologies have undeniably changed the scientific landscape in biology
In addition to libraries for sequencing purposes, many proteome-wide functional assays, for instance assessing protein interactions [2,3], protein localization [4], posttranscriptional regulation [5] or drug activity [6], rely on pooled or arrayed nucleic acid libraries as input. Some of these libraries can be accurately synthesized at relatively low cost, or one can rely on available collections of full-length and validated open reading frames (ORFs) on plasmids [7], short hairpin or small interfering RNA libraries [8] and guide RNA libraries for CRISPR screens [9]
Most molecular manipulations during library preparation introduce some form of bias, resulting in a skewed representation of the original molecules

Summary

Introduction

Generation sequencing technologies have undeniably changed the scientific landscape in biology. In addition to libraries for sequencing purposes, many proteome-wide functional assays, for instance assessing protein interactions [2,3], protein localization [4], posttranscriptional regulation [5] or drug activity [6], rely on pooled or arrayed nucleic acid libraries as input Some of these libraries can be accurately synthesized at relatively low cost, or one can rely on available collections of full-length and validated open reading frames (ORFs) on plasmids [7], short hairpin or small interfering RNA libraries [8] and guide RNA libraries for CRISPR screens [9]. Coding sequence fragment libraries are a prominent example [10,11,12,13]

Objectives

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Conclusion