The effects of a globin blocker on the resolution of 3’mRNA sequencing data in porcine blood

Abstract

Background : Gene expression profiling in blood is a potential source of biomarkers to evaluate or predict phenotypic differences between pigs but is expensive and inefficient because of the high abundance of globin mRNA in porcine blood. These limitations can be overcome by the use of 3’mRNA sequencing combined with a method to deplete or block the processing of globin mRNA prior to or during library construction. Here, we validated the effectiveness of a novel specific globin blocker (GB) that is included in the library preparation step of 3’mRNA sequencing. Results : Four concentrations of the GB were applied to RNA samples from two pigs. The GB significantly reduced the proportion of globin reads ( P = 0.005) and increased the number of detectable non-globin genes. The highest evaluated concentration (C1) of the GB resulted in the largest reduction of globin reads (from 56.4 to 10.1%). The second highest concentration C2, showed very similar globin depletion rates (12 %) as C1 but a better correlation of the expression of non-globin genes between GB and non-GB ( r = 0.98), and allowed the expression of an additional 1,295 non-globin genes to be detected. Concentration C2 was applied in the rest of the study. The distribution of the percentage of globin reads for non-GB (n=184) and GB (n=189) samples clearly showed the effects of the GB on reducing globin reads, in particular for HBB . The proportion of globin reads that remained in GB samples was found to be positively correlated with reticulocyte count of the blood sample ( P < 0.001). Conclusions : The GB for 3’mRNA sequencing is a useful tool in the quantification of whole gene expression profiles in porcine blood. The GB reduced the proportion of globin reads, thereby increasing the efficiency of sequencing non-globin mRNA. The evaluated GB method has as additional advantage that it does not require an additional step prior to or during library creation.