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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

Increased gene coverage as a result of globin reduction. GR treatment increased the detection of expressed genes (read >5). 6 high (RIN ≥7) and 2 moderate (5 ≤ RIN <7) RIN samples were sequenced in one lane and 4 low (RIN <5) RIN samples were sequenced in another lane. a) Comparisons of number of expressed genes in pre- and post-GR treatment. b) RIN influence on identifying additional genes post-GR treatment.
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Fig4: Increased gene coverage as a result of globin reduction. GR treatment increased the detection of expressed genes (read >5). 6 high (RIN ≥7) and 2 moderate (5 ≤ RIN <7) RIN samples were sequenced in one lane and 4 low (RIN <5) RIN samples were sequenced in another lane. a) Comparisons of number of expressed genes in pre- and post-GR treatment. b) RIN influence on identifying additional genes post-GR treatment.

Mentions: The number of detected genes (read counts >5) in post-GR samples was significantly increased compared to pre-GR samples (paired t-test) (Figure 4a). GR treatment increased the gene detection rate by 8.6% in high RIN samples, 2.2% in moderate and 5.4% in low RIN samples. It was also noticed that the number of additional genes identified in post-GR samples was higher for samples with a high RIN (Figure 4b). It may be noted that the detection rate was higher in high RIN samples compared to low RIN samples despite being sequenced at half the depth. Pre-GR, an average of 93 genes were uniquely detected in the high RIN group, whereas 243 genes were uniquely detected in the moderate/low RIN group. Post-GR, the corresponding uniquely detected genes in the two groups were 1,157 and 753, respectively (Additional file 1: Figure S4).Figure 4


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)

Increased gene coverage as a result of globin reduction. GR treatment increased the detection of expressed genes (read >5). 6 high (RIN ≥7) and 2 moderate (5 ≤ RIN <7) RIN samples were sequenced in one lane and 4 low (RIN <5) RIN samples were sequenced in another lane. a) Comparisons of number of expressed genes in pre- and post-GR treatment. b) RIN influence on identifying additional genes post-GR treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Increased gene coverage as a result of globin reduction. GR treatment increased the detection of expressed genes (read >5). 6 high (RIN ≥7) and 2 moderate (5 ≤ RIN <7) RIN samples were sequenced in one lane and 4 low (RIN <5) RIN samples were sequenced in another lane. a) Comparisons of number of expressed genes in pre- and post-GR treatment. b) RIN influence on identifying additional genes post-GR treatment.
Mentions: The number of detected genes (read counts >5) in post-GR samples was significantly increased compared to pre-GR samples (paired t-test) (Figure 4a). GR treatment increased the gene detection rate by 8.6% in high RIN samples, 2.2% in moderate and 5.4% in low RIN samples. It was also noticed that the number of additional genes identified in post-GR samples was higher for samples with a high RIN (Figure 4b). It may be noted that the detection rate was higher in high RIN samples compared to low RIN samples despite being sequenced at half the depth. Pre-GR, an average of 93 genes were uniquely detected in the high RIN group, whereas 243 genes were uniquely detected in the moderate/low RIN group. Post-GR, the corresponding uniquely detected genes in the two groups were 1,157 and 753, respectively (Additional file 1: Figure S4).Figure 4

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