Dec 19, 2013

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

BioRxiv : the Preprint Server for Biology
Jeanette Baran-GalePraveen Sethupathy


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 ...Continue Reading

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Mentioned in this Paper

Small Nuclear RNA
RNA library
Sequence Determinations, RNA
Research Personnel
Nucleic Acid Sequencing
tris(4,7-diphenyl-1,10-phenanthroline)ruthenium (II)
Molecular Genetic Technique

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