Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples

BMC Genomics

Le Luo Guan¹  Joan K Lunney⁴  Samuel M Abrams⁴  Eric Fritz-Waters³  James M Reecy³  Christopher K Tuggle³  Graham S Plastow¹  Paul Stothard¹  Yan Meng¹  Xu Sun¹  Afshin Hosseini²  Arun Kommadath¹  Hua Bao¹  Igseo Choi⁴ 

[1] Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada;Present address: Institute for Animal Science, Physiology and Hygiene Unit, University of Bonn, Katzenburgweg 7-9, 53115 Bonn, Germany;Department of Animal Science, Iowa State University, Ames, IA, USA;Animal Parasitic Diseases Laboratory, ARS, USDA, Beltsville, MD, USA

关键词: Transcriptome;    RNA-seq;    Globin reduction;    Blood;    Pig;   

Others  :  1127736
DOI  :  10.1186/1471-2164-15-954

 received in 2014-10-01, accepted in 2014-10-16,  发布年份 2014

【 摘 要 】

Background

Transcriptome analysis of porcine whole blood has several applications, which include deciphering genetic mechanisms for host responses to viral infection and vaccination. The abundance of alpha- and beta-globin transcripts in blood, however, impedes the ability to cost-effectively detect transcripts of low abundance. Although protocols exist for reduction of globin transcripts from human and mouse/rat blood, preliminary work demonstrated these are not useful for porcine blood Globin Reduction (GR). Our objectives were to develop a porcine specific GR protocol and to evaluate the GR effects on gene discovery and sequence read coverage in RNA-sequencing (RNA-seq) experiments.

Results

A GR protocol for porcine blood samples was developed using RNase H with antisense oligonucleotides specifically targeting porcine hemoglobin alpha (HBA) and beta (HBB) mRNAs. Whole blood samples (n = 12) collected in Tempus tubes were used for evaluating the efficacy and effects of GR on RNA-seq. The HBA and HBB mRNA transcripts comprised an average of 46.1% of the mapped reads in pre-GR samples, but those reads reduced to an average of 8.9% in post-GR samples. Differential gene expression analysis showed that the expression level of 11,046 genes were increased, whereas 34 genes, excluding HBA and HBB, showed decreased expression after GR (FDR <0.05). An additional 815 genes were detected only in post-GR samples.

Conclusions

Our porcine specific GR primers and protocol minimize the number of reads of globin transcripts in whole blood samples and provides increased coverage as well as accuracy and reproducibility of transcriptome analysis. Increased detection of low abundance mRNAs will ensure that studies relying on transcriptome analyses do not miss information that may be vital to the success of the study.

【 授权许可】

   
2014 Choi et al.; licensee BioMed Central Ltd.

【 图 表 】

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