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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

Venn diagram of number of genes identified as expressed in pre- versus post-GR samples. There is significant overlap (11,773) between pre- and post-GR samples. An additional 815 genes were identified in only post-GR samples.
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Fig5: Venn diagram of number of genes identified as expressed in pre- versus post-GR samples. There is significant overlap (11,773) between pre- and post-GR samples. An additional 815 genes were identified in only post-GR samples.

Mentions: We next determined genes expressed in porcine whole blood using all 12 samples, based on the criterion that a gene was detected at read counts above 5 in at least 5 of the 12 samples. We identified 12,588 genes in post-GR samples and 11,826 genes in pre-GR samples with an overlap of 11,773 genes (Figure 5). Excluding the overlap, 815 genes were detected only in post-GR samples, whereas 53 were specific to pre-GR samples. The small number of genes found specific to pre-GR samples may be due to the effect of RIN or technical variations. A comparison of the mean expressions of the set of 11,773 genes detected in both pre- and post-GR samples and the 815 genes detected only in post-GR samples revealed increased expression in post-GR samples (Additional file 1: Figure S5). The mean expression of the 815 additional genes in post-GR samples was well below the lower quartile of the expression levels of genes common to both pre- and post-GR samples. Thus GR treatment increases the ability to detect genes expressed at very low levels.Figure 5


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)

Venn diagram of number of genes identified as expressed in pre- versus post-GR samples. There is significant overlap (11,773) between pre- and post-GR samples. An additional 815 genes were identified in only post-GR samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Venn diagram of number of genes identified as expressed in pre- versus post-GR samples. There is significant overlap (11,773) between pre- and post-GR samples. An additional 815 genes were identified in only post-GR samples.
Mentions: We next determined genes expressed in porcine whole blood using all 12 samples, based on the criterion that a gene was detected at read counts above 5 in at least 5 of the 12 samples. We identified 12,588 genes in post-GR samples and 11,826 genes in pre-GR samples with an overlap of 11,773 genes (Figure 5). Excluding the overlap, 815 genes were detected only in post-GR samples, whereas 53 were specific to pre-GR samples. The small number of genes found specific to pre-GR samples may be due to the effect of RIN or technical variations. A comparison of the mean expressions of the set of 11,773 genes detected in both pre- and post-GR samples and the 815 genes detected only in post-GR samples revealed increased expression in post-GR samples (Additional file 1: Figure S5). The mean expression of the 815 additional genes in post-GR samples was well below the lower quartile of the expression levels of genes common to both pre- and post-GR samples. Thus GR treatment increases the ability to detect genes expressed at very low levels.Figure 5

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