Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

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. Studies have shown that adapter ligation introduces a significant but widely unappreciated bias in the results of high-throughput small RNA sequencing. We show that due to this bias the two widely used Illumina library preparation protocols produce strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that are >5-fold differentially detected and some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. 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. In an attempt to mitigate this bias, the new Bioo Scientific NEXTflex V2 protocol utilizes a pool of adapters with random nucleotides at the ligation boundary. We show that this protocol is able to detect robustly several miRNAs that evade capture by the Illumina-based methods. While these analyses do not indicate a definitive gold standard for small RNA library preparation, the results of the NEXTflex protocol do correlate best with RT-qPCR. As increasingly more laboratories seek to study small RNAs, researchers should be aware of the extent to which the results may differ with different protocols, and should make an informed decision about the protocol that best fits their study.

Cajal body function in genome organization and transcriptome diversity

“…However, CBs, and NBs in general, influence many more processes than transcription and future experiments across a range of cell types with different transcriptional profiles will be critical to understand how this diversity is generated. Improvements to next-generation sequencing tools for the detection and characterization of small RNAs indicates that the future holds a great deal of promise for dissecting the multiple roles of CBs in RNA transcription, processing [131, 132], RNA base modification [133], and UTR length variation of specific genes [134]. …”

Sex-biased microRNA expression in mammals and birds reveals underlying regulatory mechanisms and a role in dosage compensation

“…To mitigate the extensive sequencing biases that have been identified for standard library preparation methods (Sorefan et al 2012;Baran-Gale et al 2015), we chose to prepare the libraries with the NEXTflex Small RNA-seq Kit (Methods), which improves the ligation step through the use of partially degenerate adapters (Baran-Gale et al 2015). We found that this protocol allowed substantially broader coverage of expressed miRNAs compared to standard methods (Supplemental Fig.…”

Toward the promise of microRNAs – Enhancing reproducibility and rigor in microRNA research

“…[28][29][30] Two studies have demonstrated wide differences in microRNA RPM depending on the use of different library preparation kits (TruSeq small RNA [Illumina], NEXTflex [Bioo Scientific], and NEBNext [New England Biolabs]), with some microRNAs having counts as high as 100,000 RPM by one method and <1,000 RPM by a different method from matched samples. 31,32 Thus in comparing studies, it is important to keep these concerns in mind and for all projects within a given laboratory or core sequencing facility be consistent in regards to the preparation method used.…”