Researchers from the University of North Carolina at Chapel Hill, the National Human Genome Research Institute, and the NIH Intramural Sequencing Center, recently published their research, "Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods," in the journal Frontiers in Genetics. Their research supports previously published works indicating that the adapter ligation steps of library prep introduce a significant but widely unappreciated bias into the results of high-throughput small RNA sequencing.
Baran-Gale and others isolated total RNA from mouse insulinoma (MIN6) cells and constructed small RNA libraries using the Illumina v1.5 protocol and the Illumina TruSeq protocol. Results obtained with the two widely used Illumina library preparation protocols produced strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that were >5-fold differentially detected and some miRNAs, such as miR-24-3p, were over 30-fold differentially detected. While some level of bias in library preparation was not surprising to the researchers, the apparent massive differential bias between these two widely used adapter sets was not well appreciated.
The researchers then used the NEXTflex Small RNA-Seq Kit v2, which utilizes adapters with random nucleotides at the ligation boundary, to prepare libraries from the same total RNA. Their results indicated that the NEXTflex Small RNA-Seq Kit v2 protocol was able to robustly detect several miRNAs that partially or completely evaded capture by the Illumina-based methods. These results also correlated best with RT-qPCR data.
Baran-Gale, J., Kurtz, L. C., Erdos, Sison, M. C., Young, A., Fannin, E. E., Chines, P. S. and Sethupathy, P. (2015) Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods. Frontiers in Genetics. doi: 10.3389/fgene.2015.00352.