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

Choi I, Bao H, Kommadath A, Hosseini A, Sun X, Meng Y, Stothard P, Plastow GS, Tuggle CK, Reecy JM, Fritz-Waters E, Abrams SM, Lunney JK, Guan le L - BMC Genomics (2014)

Bottom Line: 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).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).

View Article: PubMed Central - PubMed

Affiliation: Animal Parasitic Diseases Laboratory, ARS, USDA, Beltsville, MD, USA. Joan.Lunney@ars.usda.gov.

ABSTRACT

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.

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Related in: MedlinePlus

Normalized 5′ to 3′ sequence coverage by position showing lower expression in one representative low RIN sample. For lower expressed 4,792 genes in low RIN samples, the average read numbers of low RIN (<5) and moderate/high RIN (≥5) in pre- and post-GR samples is shown at each relative position.
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Fig3: Normalized 5′ to 3′ sequence coverage by position showing lower expression in one representative low RIN sample. For lower expressed 4,792 genes in low RIN samples, the average read numbers of low RIN (<5) and moderate/high RIN (≥5) in pre- and post-GR samples is shown at each relative position.

Mentions: Figure 2b depicts a heatmap of the normalized log2 transformed expression of the 11,046 genes with higher level of detection in post-GR samples compared to pre-GR samples. It was observed that a large set of genes in the low RIN samples (within the boxes in Figure 2b) was considerably lower expressed than the corresponding set in the high/moderate RIN samples, both pre- and post-GR. We believe that these are the genes with the greatest degradation in the low RIN samples. We then examined the variation in gene body coverage from 5′ to 3′ in high/moderate and low RIN samples, respectively. Low RIN samples showed strong bias toward increased coverage at the 3′ end (Figure 3). Among the low-quality RNA samples, pre- and post-GR treatment showed the same trend of bias, which affirmed that the RNase H treatment was not the determining factor. High quality samples showed better coverage from 5′ to 3′ as well as at the ends in both pre- and post-GR treated samples. All low quality samples were biased toward increased coverage at the 3′ end, possibly due to the degradation of RNA. However, the number of unique genes detected did not differ significantly between low and high RIN samples.Table 3


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

Choi I, Bao H, Kommadath A, Hosseini A, Sun X, Meng Y, Stothard P, Plastow GS, Tuggle CK, Reecy JM, Fritz-Waters E, Abrams SM, Lunney JK, Guan le L - BMC Genomics (2014)

Normalized 5′ to 3′ sequence coverage by position showing lower expression in one representative low RIN sample. For lower expressed 4,792 genes in low RIN samples, the average read numbers of low RIN (<5) and moderate/high RIN (≥5) in pre- and post-GR samples is shown at each relative position.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230834&req=5

Fig3: Normalized 5′ to 3′ sequence coverage by position showing lower expression in one representative low RIN sample. For lower expressed 4,792 genes in low RIN samples, the average read numbers of low RIN (<5) and moderate/high RIN (≥5) in pre- and post-GR samples is shown at each relative position.
Mentions: Figure 2b depicts a heatmap of the normalized log2 transformed expression of the 11,046 genes with higher level of detection in post-GR samples compared to pre-GR samples. It was observed that a large set of genes in the low RIN samples (within the boxes in Figure 2b) was considerably lower expressed than the corresponding set in the high/moderate RIN samples, both pre- and post-GR. We believe that these are the genes with the greatest degradation in the low RIN samples. We then examined the variation in gene body coverage from 5′ to 3′ in high/moderate and low RIN samples, respectively. Low RIN samples showed strong bias toward increased coverage at the 3′ end (Figure 3). Among the low-quality RNA samples, pre- and post-GR treatment showed the same trend of bias, which affirmed that the RNase H treatment was not the determining factor. High quality samples showed better coverage from 5′ to 3′ as well as at the ends in both pre- and post-GR treated samples. All low quality samples were biased toward increased coverage at the 3′ end, possibly due to the degradation of RNA. However, the number of unique genes detected did not differ significantly between low and high RIN samples.Table 3

Bottom Line: 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).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).

View Article: PubMed Central - PubMed

Affiliation: Animal Parasitic Diseases Laboratory, ARS, USDA, Beltsville, MD, USA. Joan.Lunney@ars.usda.gov.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus