Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNA
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METHODOLOGY ARTICLE
Open Access
Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs Jin Sung Jang1,2* , Brianna Berg1, Eileen Holicky1, Bruce Eckloff1, Mark Mutawe1, Minerva M. Carrasquillo3, Nilüfer Ertekin-Taner3,4 and Julie M. Cuninngham1,2*
Abstract Background: There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 μg of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated with DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. Results: Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3′ region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R > 0.9) than RNAse-H enzymatic depletion (R > 0.85). Conclusion: In this study, our results showed that 1 μg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures. Keywords: mRNA-Seq, Globin gene depletion, rRNA, Whole blood
* Correspondence: [email protected]; [email protected] 1 Medical Genome Facility, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's
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