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Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing

Jeanette Baran-Gale, Michael R. Erdos, Christina Sison, Alice Young, Emily E. Fannin, Peter S. Chines, Praveen Sethupathy
doi: https://doi.org/10.1101/001479
Jeanette Baran-Gale
1Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
2Bioinformatics and Computational Biology Curriculum, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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Michael R. Erdos
3National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892
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Christina Sison
4NIH Intramural Sequencing Center, National Institutes of Health, Rockville, MD 20852
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Alice Young
4NIH Intramural Sequencing Center, National Institutes of Health, Rockville, MD 20852
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Emily E. Fannin
1Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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Peter S. Chines
3National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892
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Praveen Sethupathy
1Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
2Bioinformatics and Computational Biology Curriculum, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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  • For correspondence: praveen_sethupathy@med.unc.edu
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Abstract

Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of adapter sequences to both the 5’ and 3’ ends of small RNAs. Two widely used protocols for small RNA library preparation, Illumina v1.5 and Illumina TruSeq, use different pairs of adapter sequences. In this study, we compare the results of small RNA-sequencing between v1.5 and TruSeq and observe a striking differential bias. Nearly 100 highly expressed microRNAs (miRNAs) are >5-fold differentially detected and 48 miRNAs are >10-fold differentially detected between the two methods of library preparation. In fact, some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. The results are reproducible across different sequencing centers (NIH and UNC) and both major Illumina sequencing platforms, GAIIx and HiSeq. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. As increasingly more laboratories transition to the newer TruSeq-based library preparation for small RNAs, researchers should be aware of the extent to which the results may differ from previously published results using v1.5.

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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-NC-ND Unported 3.0 license.
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Posted December 19, 2013.
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Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing
Jeanette Baran-Gale, Michael R. Erdos, Christina Sison, Alice Young, Emily E. Fannin, Peter S. Chines, Praveen Sethupathy
bioRxiv 001479; doi: https://doi.org/10.1101/001479
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Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing
Jeanette Baran-Gale, Michael R. Erdos, Christina Sison, Alice Young, Emily E. Fannin, Peter S. Chines, Praveen Sethupathy
bioRxiv 001479; doi: https://doi.org/10.1101/001479

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