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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Publications at this Location » Publication #299683

Research Project: FUNCTIONAL GENOMIC APPROACHES FOR CONTROLLING DISEASES OF SWINE

Location: Animal Parasitic Diseases Laboratory

Title: RNA sequencing for increasing gene discovery and and coverage using globin RNA reduced porcine blood samples

Author
item Choi, Igseo
item BAO, H - University Of Alberta
item KOMMADATH, A - University Of Alberta
item HOSSEINI, A - University Of Alberta
item SUN, X - University Of Alberta
item MENG, Y - University Of Alberta
item STOTHARD, P - University Of Alberta
item PLASTOW, G - University Of Alberta
item TUGGLE, C - Iowa State University
item REECY, J - Iowa State University
item FRITZ, E - Iowa State University
item Abrams, Samuel
item Lunney, Joan
item GUAN, L - University Of Alberta

Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/16/2014
Publication Date: 10/17/2014
Citation: Choi, I., Bao, H., Kommadath, A., Hosseini, A., Sun, X., Meng, Y., Stothard, P., Plastow, G., Tuggle, C.K., Reecy, J.M., Fritz, E., Abrams, S.M., Lunney, J.K., Guan, L. 2014. RNA sequencing for increasing gene discovery and and coverage using globin RNA reduced porcine blood samples. Biomed Central (BMC) Genomics. 15:954.

Interpretive Summary: A major goal of our research with Porcine Reproductive and Respiratory Syndrome (PRRS) is to decipher genetic mechanisms controlling host responses to viral infection. To address this we have analyzed blood samples from PRRS virus infected pigs as part of our PRRS Host Genetic Consortium studies. The abundance of porcine globin gene transcripts, however, impedes the ability to detect less abundant transcripts. Gene expression analyses were performed with next generation RNA-seq technology using Illumina HiScan SQ system. The objective of this study was to develop and evaluate porcine specific globin reduction protocols using globin gene (HBA and HBB) oligonucleotides and RNA-seq. Porcine whole blood was preserved in Tempus tubes and RNAs prepared. The cDNA libraries were made from control and post globin reduction treatment RNAs. Our results confirmed that after globin reduction treatment HBA and HBB mRNAs were efficiently depleted (90% and 89%, respectively). This resulted in increased detection of novel genes, i.e., compared to control samples, 11,046 genes showed higher expression and 34 genes, excluding HBA and HBB, showed lower expression after globin reduction treatment.In addition, 1,030 genes on average were newly identified after globin reduction. In conclusion, our porcine specific globin reduction protocol minimized the number of reads of globin RNA and provided increased accuracy and reproducibility of transcriptome analysis. Based on these results we will now be able to unravel the complexities of host response to Porcine Reproductive and Respiratory Syndrome virus infection.

Technical Abstract: Background Transcriptome analysis in porcine whole blood will provide major insights to decipher genetic mechanisms for host responses to viral infection. The abundance of porcine globin transcripts, however, impedes the ability to detect less abundant transcripts. The objective of our study was to develop and evaluate a porcine specific globin reduction protocol using globin (HBA and HBB) oligonucleotides and RNA-seq. Results Whole blood samples were collected in Tempus tubes and RNAs prepared. The cDNA libraries were made from control and post globin reduction RNAs. RNA-seq analyses were performed using the Illumina HiScan SQ system. The uniquely mapped reads of HBA and HBB tags comprised up to 25% of the pre-treatment reads; this was reduced to 0.1 - 2.5% post globin RNA reduction. For validation, qPCR results confirmed that HBA and HBB mRNAs were efficiently depleted (90% and 89%, respectively). Compared to control samples, 11,046 genes showed higher expression and 34 genes, excluding HBA and HBB, showed lower expression after globin reduction treatment (5% FDR level). Conclusions In conclusion, our porcine specific globin reduction protocol minimizes the number of reads of globin RNA and provides increased accuracy and reproducibility of transcriptome analysis. Based on these results we will now be able to unravel the complexities of host response to Porcine Reproductive and Respiratory Syndrome virus infection.