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Beyond library size: a field guide to NGS normalization

View ORCID ProfileJelena Aleksic, View ORCID ProfileSarah Carl, Michaela Frye
doi: https://doi.org/10.1101/006403
Jelena Aleksic
1Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom
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  • For correspondence: j.aleksic@gen.cam.ac.uk s.carl@gen.cam.ac.uk mf364@cam.ac.uk
Sarah Carl
2Department of Genetics and Cambridge Systems Biology Centre, University of Cambridge, Downing Street, Cambridge CB2 3EH, United Kingdom
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Michaela Frye
1Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom
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Abstract

Background: Next generation sequencing (NGS) is a widely used technology in both basic research and clinical settings and it will continue to have a major impact on biomedical sciences. However, the use of incorrect normalization methods can lead to systematic biases and spurious results, making the selection of an appropriate normalization strategy a crucial and often overlooked part of NGS analysis.

Results: We present a basic introduction to the currently available normalization methods for differential expression and ChIP-seq applications, along with best use recommendations for different experimental techniques and datasets.

We demonstrate that the choice of normalization technique can have a significant impact on the number of genes called as differentially expressed in an RNA-seq experiment or peaks called in a ChIP-seq experiment.

Conclusions: The choice of the most adequate normalization method depends on both the distribution of signal in the dataset and the intended downstream applications. Depending on the design and purpose of the study, appropriate bias correction should also be considered.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted June 19, 2014.
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Beyond library size: a field guide to NGS normalization
Jelena Aleksic, Sarah Carl, Michaela Frye
bioRxiv 006403; doi: https://doi.org/10.1101/006403
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Beyond library size: a field guide to NGS normalization
Jelena Aleksic, Sarah Carl, Michaela Frye
bioRxiv 006403; doi: https://doi.org/10.1101/006403

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