Limits...
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.

Show MeSH

Related in: MedlinePlus

Differential gene expression in pre- versus post-GR samples. a. MA plot revealed that the majority of the differentially expressed genes showed increased detection (11,046) in post-GR samples and only 34 (excluding globin genes) showed lower detection. b. Heatmap shows the expression profile across all samples for the 11,046 genes with increased detection by GR treatment. A large set of genes in the low RIN samples (within the orange boxes) was considerably lower expressed than the corresponding set in the high/moderate RIN samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Differential gene expression in pre- versus post-GR samples. a. MA plot revealed that the majority of the differentially expressed genes showed increased detection (11,046) in post-GR samples and only 34 (excluding globin genes) showed lower detection. b. Heatmap shows the expression profile across all samples for the 11,046 genes with increased detection by GR treatment. A large set of genes in the low RIN samples (within the orange boxes) was considerably lower expressed than the corresponding set in the high/moderate RIN samples.

Mentions: Following an approach similar to that described by Mastrokolias et al. [3], we investigated the effect of GR on enhancing the coverage of non-globin genes and the sensitivity of gene expression detection. Read count data was normalized by library size and DE genes between pre- and post-GR samples were determined using edgeR (see Methods). Compared to pre-GR samples, 11,046 genes showed higher level of detection (expression) and 34 genes (Table 3), excluding HBA and HBB and ENSSSCG00000014727 (hemoglobin subunit beta-like), showed lower level of detection after GR treatment (FDR <0.05) (Figure 2a). We checked for sequence similarities among these 34 genes and the four globin oligonucleotides for possible non-target specific hybridization, but found none.


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)

Differential gene expression in pre- versus post-GR samples. a. MA plot revealed that the majority of the differentially expressed genes showed increased detection (11,046) in post-GR samples and only 34 (excluding globin genes) showed lower detection. b. Heatmap shows the expression profile across all samples for the 11,046 genes with increased detection by GR treatment. A large set of genes in the low RIN samples (within the orange boxes) was considerably lower expressed than the corresponding set in the high/moderate RIN samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Differential gene expression in pre- versus post-GR samples. a. MA plot revealed that the majority of the differentially expressed genes showed increased detection (11,046) in post-GR samples and only 34 (excluding globin genes) showed lower detection. b. Heatmap shows the expression profile across all samples for the 11,046 genes with increased detection by GR treatment. A large set of genes in the low RIN samples (within the orange boxes) was considerably lower expressed than the corresponding set in the high/moderate RIN samples.
Mentions: Following an approach similar to that described by Mastrokolias et al. [3], we investigated the effect of GR on enhancing the coverage of non-globin genes and the sensitivity of gene expression detection. Read count data was normalized by library size and DE genes between pre- and post-GR samples were determined using edgeR (see Methods). Compared to pre-GR samples, 11,046 genes showed higher level of detection (expression) and 34 genes (Table 3), excluding HBA and HBB and ENSSSCG00000014727 (hemoglobin subunit beta-like), showed lower level of detection after GR treatment (FDR <0.05) (Figure 2a). We checked for sequence similarities among these 34 genes and the four globin oligonucleotides for possible non-target specific hybridization, but found none.

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