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Bianka Baying, Bettina Haase, Jonathon Blake, Dinko Pavlinic, Jürgen Zimmermann, Vladimir Benes EMBL-GeneCore |
Abstract
Next generation sequencing (NGS) offers an unprecedented opportunity to characterize comprehensively components of the entire cellular nucleic acids content, including its modifications such as cytosine methylation, for example, and RNA or DNA sequences bound by their cognate proteins. However, with the exception of genomic DNA, whose content in the cell is more or less constant (apart from amplifications associated with various pathological conditions), nucleic acid levels in the cell are highly variable due to cell state dynamics. The complexity is furthermore compound by the variations in source, amount and quality of nucleic acid under investigation.
So far there is no NGS system that enables us to explore individual cellular nucleic acid components without a ‘pre-processing’ step, i.e. preparation of sequencing library. Ideally, each library should reflect sample complexity without missing anything. However, variable abundance of investigated nucleic acid molecules, their uneven base composition, inefficiencies of in vitro reactions or unrecognized sequence preferences of used enzymes make this goal hard to achieve. Even the way RNA molecules are fragmented impacts on the representation of particular transcripts in the obtained data. Introduction of magnetic beads, developments in enzymology and miniaturization of library preparation protocols have considerably improved our capability to prepare good sequencing libraries, resulting in certain applications even in the omission of a PCR step. The market place continues bringing out new applications and protocols to deal with issues seen in NGS library preparation. We have approached these critically when adopting new products in our workflows.
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