- Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs. - 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 μ g of whole blood-derived RNA on mRNA-Seq profiling. - 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 of the total mapped reads. - Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R >. - Conclusion: In this study, our results showed that 1 μ g 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. - 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 Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - Full list of author information is available at the end of the article. - Transcriptome profiling of peripheral whole blood sam- ples is highly desirable for biological research, drug dis- covery, diagnostic testing, and developing biomarkers in clinical settings [1–4]. - Total RNA from whole blood contains a large portion of globin genes, which originate from red blood cells and accounts for 80–90% of total transcripts [4]. - Thus, globin gene depletion is an essential step to obtain accurate data for transcriptome analysis.. - For transcriptome profiling performs in whole blood, most kits for total RNA-Seq include both rRNA and glo- bin gene depletion steps before generating the first- strand cDNA. - However, we observed significant globin and rRNA gene reads in some whole transcriptome ana- lyses of whole blood derived total RNA, suggesting that the depletion methods may be improved. - 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. - In this study, we evaluated two methods for globin gene removal, probe hybridization and RNase H-based enzymatic digestion. - The data generated from four com- mercially available kits were analyzed for performance on mRNA-Seq for whole blood-derived RNA transcrip- tome. - Our results provide information on which of the globin gene removal kit is most suitable for mRNA-Seq data analysis from whole blood samples.. - Globin-depleted total RNA samples were checked for quality on a BioAnalyser 2100 high sensitivity DNA chip for all kits. - Total RNA was extracted from six samples collected in Paxgene Blood Tubes and treated with DNaseI. - Technical replicates of 1 μ g of each sample underwent depletion with one of the four kits and sequenced using the poly-A+ selection protocols. - rRNA Depletion Kit. - Based on the RNA peaks from the electrophero- gram profile in the two enzymatic depletion kits, the NEBNext® Globin &. - For Globin-Zero Gold rRNA Removal Kit (GZr), a probe hybridization method, RNA. - The libraries generated from the four kits were se- quenced to evaluate performance, particularly the effi- ciency of the globin gene depletion, using stranded mRNA-Seq with poly-A+ selection and sequencing data are summarized in Table 1. - Total RNA depleted by the four different kits were analyzed using a Bioanalyzer 2100 High sensitivity DNA chip. - reads mapped to the genome averaged 30 million (M) reads (22 M–38 M), with exon reads at . - 0.05) in the NEBgr with 6.3% (±2.3. - 1% from the total mapped reads (Fig. - The total junction reads were signifi- cantly higher in the probe hybridization depletion method GLOBINClear and GZr (37–40% from total mapped reads, p <. - In addition, the gene body coverage plot showed that the probe hybridization method covered the en- tire gene body uniformly. - In contrast, the enzymatic removal methods revealed skewed expression to the 3′ region of genes, indicating that RNA degradation likely occurred dur- ing the depletion step (Fig. - Next, NEBgr was excluded from the second analysis be- cause of the significant quantity of transcripts from globin genes remaining in the total reads. - These samples were sequenced with aver- age 56 M - 72 M reads mapping to the genome, exon reads were similar to those in the first dataset (81.8–. - The rRNA reads were signifi- cantly higher in the GLOBINClear (p <. - 0.0001) but still below 2% from the total mapped reads (Fig. - As ob- served in the first data set, the probe hybridization method yielded more junction reads (38–39%) than enzymatic re- moval methods (31–32%, Fig. - However, at the transcript level, significantly more transcripts were detected in the GLOBINClear (85,979), with 78,526 transcripts observed in the RZr, and 82,669 transcripts in the GZr (Fig. - Stranded mRNA-Seq was used to assess four globin gene depletion kits to allow a sensitive assessment of the. - a Percentage of globin gene contamination in the total mapped reads, b Percentage of rRNA contamination in the total mapped reads, c Percentage of junction reads in the total mapped reads. - The probe hybridization method covered the entire gene body uniformly. - b Representative screenshot in the long transcript between two different depletion methods. - GLOBINClear Kit covered more reads in the middle of the gene than Ribo-Zero Plus Kit. - From exon 11 to 19 of the ATM gene were visualized on the IGV.. - Globin gene depletion from whole-blood derived RNA does reduce both the amount and quality of RNA [8] but is an essential procedure for global RNA-Seq analysis. - In this study, we directly com- pared the performances of both probe hybridization and RNAse-H enzymatic depletion methods using four com- mercially available kits using mRNA Seq. - Overall, the probe hybridization method showed a better perform- ance with an increased total number of genes and tran- scripts detected without 3′ region bias seen with the enzymatic depletion methods.. - 99% of globin genes in three of the four kits. - While residual rRNA contamination was found in all tested samples ranging from 0.2–2% level of the total mapped reads, high-quality sequencing data mapping to the reference genome at >. - 96% of the total reads was generated, significantly better than previously reported . - Among the Globin gene and rRNA removal kits, the probe hybridization method, GZr showed the lowest re- covery yields likely related to the multiple cleanup steps required to remove the rRNA and globin genes. - The RNase H-based RNA depletion, RZr, method was faster with higher recovery yields, and more streamlined pro- cessing than the probe hybridization method, with all enzymatic reactions carried out in a single tube.. - However, the combined RNase H and DNAseI enzyme activity did affect RNA quality and subsequently gener- ated 3′ biased sequencing data, particularly in the longer transcripts. - Overall, we observed that the RNase H- based RNA depletion method generated significantly fewer junction reads and a reduced number of total transcripts than the probe hybridization method. - There- fore, due to the partial degradation of mRNA during the depletion step, RNase H-based RNA depletion may be a more appropriate method for the total RNA sequencing, which does not require poly-A+ selection.. - Between the probe hybridization depletion method kits, GZr showed a reduced correlation than RZr when compared to GLOBINClear at the gene level. - We as- sume that the total input of the depleted RNA for mRNA-seq library construction affects detecting the ex- pression level of the lower copy of genes and transcripts between two kits. - this may be the main cause of reduced correlation at the gene level between two kits as GZr tends to lose RNA during cleanup of the hybridized streptavidin beads. - Subsequently, poly-A se- lection is required at the beginning of the mRNA-Seq li- brary construction procedure, which is a double negative selection of rRNA in the GZr group. - However, as a re- sult, GZr showed the best performance of the depletion of both rRNA and Globin genes from the total mapped reads. - Thus, the probe hybridization depletion is an appropri- ate method for the mRNA sequencing that is both. - In this study, we showed 1 μg of high-quality RNA from whole blood collected in PAXgene Blood RNA tubes may be routinely used for transcriptional profiling ana- lysis studies. - In addition, we have demonstrated that the probe hybridization depletion method is more suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures from whole blood- derived RNA. - Therefore, our results should help bio- banking efforts that allow us to do more affordable mRNA sequencing with high resolution of transcriptome profile study of whole blood.. - Total RNA extraction from whole blood. - Peripheral whole blood samples from six volunteers were collected in PAXgene Blood RNA tubes (PreAn- alytiX GmbH, BD Biosciences, Mississauga, ON, Canada) following institutionally approved IRBs. - Total RNA was extracted from four aliquots using a PAXgene Blood RNA Kit with DNaseI treatment (Qiagen, Chats- worth, CA, USA) according to the manufacturer’s proto- col. - a The total number of detected genes, b The total number of detected transcripts, c Correlation values between samples using the total number of detected genes, d Correlation values between samples using the total number of detected transcripts. - 7.5 proceeded to the glo- bin mRNA and rRNA removal step from total RNA. - Globin gene depletion. - Washed streptavidin magnetic beads were added and incubated for 30 min, then the beads were pulled to the bottom of the plate on a magnet plate, and globin- depleted mRNA was transferred to a new tube. - Another 1 μg of total RNA was used for both globin mRNA and rRNA (cytoplasmic and mitochondrial) depletion using NEBNext® Globin &. - rRNA Depletion Kit (NEBgr) (NEB, MA, USA), Ribo-Zero Plus rRNA Depletion Kit (RZr) (Illumina, CA, USA), or Globin-Zero Gold rRNA Re- moval Kit (GZr) (Illumina, CA, USA), according to the manufacturer’s protocols with some modifications. - For GZr, total RNA was mixed with biotinylated oligonucle- otides mixture, and the streptavidin magnetic beads were added and incubated for binding to the hybridized mix- ture. - The streptavidin magnetic beads were pulled down to the bottom of the plate on a magnet plate, and the depleted RNA was transferred to the new plate for the next RNA cleanup step. - Briefly, total RNA was hybridized with single-strand DNA probes, and RNase H digestion was performed. - mRNA-Seq library construction and data analysis. - The TrueSeq Stranded mRNA-Seq Kit (Illumina, CA, USA) with poly-A+ selection was used to generate the mRNA-Seq library from depleted RNA samples using four different kits, according to the manufacturer’s protocol. - The STAR (2.6.1d) aligner was used to align reads to the human reference genome (hg38). - Also, the BAM files were used for globin gene and rRNA quantification, calculated using the percentage of the total mapped reads using Partek E/M al- gorithm. - Sequence mapping to the genome and the transcriptome was visualized in Integrative Genomics Viewer (IGV, Broad Institute). - Pearson r value was used for correlation analysis of mRNA-Seq data from each kit.. - mRNA-Seq: mRNA sequencing. - Eric Wieben for critical review and helpful revision to the manuscript. - We also thank members of the Genome Analysis Core for technical support.. - The sequencing datasets analyzed during the current study were deposited in the GEO repository (GSE150587, http://www.ncbi.nlm.nih.gov/geo/query/. - A whole-blood transcriptome meta-analysis identifies gene expression signatures of cigarette smoking. - Whole blood transcriptome analysis in amyotrophic lateral sclerosis: a biomarker study. - Whole blood transcriptome analysis reveals potential competition in metabolic pathways between negative energy balance and response to inflammatory challenge. - Whole blood RNA sequencing reveals a unique transcriptomic profile in patients with ARDS following hematopoietic stem cell transplantation. - Effects of globin mRNA reduction methods on gene expression profiles from whole blood. - Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion
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