RT Journal Article
SR Electronic
T1 Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 001479
DO 10.1101/001479
A1 Jeanette Baran-Gale
A1 Michael R. Erdos
A1 Christina Sison
A1 Alice Young
A1 Emily E. Fannin
A1 Peter S. Chines
A1 Praveen Sethupathy
YR 2013
UL http://biorxiv.org/content/early/2013/12/19/001479.abstract
AB 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.