Abstract
BackgroundThere 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.ResultsProbe 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).ConclusionIn 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.
Highlights
There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and Ribosomal RNA (rRNA) are frequent contaminants
We observed significant globin and rRNA gene reads in some whole transcriptome analyses of whole blood derived total RNA, suggesting that the depletion methods may be improved. mRNA library preparation kits do not include rRNA or globin depletion as selection of poly-A+ RNA enriches for protein-coding genes and overall it is a more costeffective and sensitive approach for gene quantification and their biological function and roles when this is the primary research goal [11]
Our results provide information on which of the globin gene removal kit is most suitable for mRNA-Seq data analysis from whole blood samples
Summary
There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. MRNA library preparation kits do not include rRNA or globin depletion as selection of poly-A+ RNA enriches for protein-coding genes and overall it is a more costeffective and sensitive approach for gene quantification and their biological function and roles when this is the primary research goal [11]. For this reason, we used stranded mRNA-Seq to evaluate globin gene removal to assess the quality of globin-depleted RNA to quantify gene expression. The evaluation will inform RNA preparation for mRNA sequencing applications
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