Globin Reduction in Porcine Whole Blood for Improving Sensitivity and Accuracy of Transcriptome Analysis

Transcriptome analysis in porcine whole blood will provide major insights to decipher genetic mechanisms for host response 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. Whole blood samples were collected in PAXgene (n = 6) and Tempus (n = 17) tubes. RNA-seq was performed for pre/post globin reduction treatment samples using Illumina HiScan SQ system. On average, 46.9 and 35.6 million reads were generated and 36.1 and 27.7 million reads were uniquely mapped in pre versus post globin reduction treated samples, respectively. The uniquely mapped reads of HBA and HBB tags comprised up to 55% 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 (97.2% and 96%, respectively). In addition, 1,030 genes on average were newly identified after globin reduction, which are annotated with relevant biological functions including several canonical immune pathways. 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. This work was supported by funds from Genome Alberta, Genome Canada, and USDA ARS and NIFA.