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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

The average proportions ofHBAandHBBreads to total mapped reads in pre- and post-GR samples. Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of HBA and HBB reads to mapped reads significantly (Paired Wilcoxon signed rank test, p <0.001).
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Fig1: The average proportions ofHBAandHBBreads to total mapped reads in pre- and post-GR samples. Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of HBA and HBB reads to mapped reads significantly (Paired Wilcoxon signed rank test, p <0.001).

Mentions: More than 653 million (M) sequence reads generated from 12 pre- and post-GR samples passed Illumina’s CASAVA (v.1.8) filtration (Table 2). These reads were then aligned to the pig genome build 10.2 by TopHat (v. 2.0.8). After GR treatment, total filtered reads and mapped reads were reduced by an average of 6.1 M and 6.8 M reads, respectively, and globin reads were reduced by an average of 11.4 M reads. The percentage of globin reads among all aligned reads averaged 46.1% and of these, 84.7% were removed by GR treatment. The proportion of globin reads to mapped reads were 46.1% and 8.9% in pre- and post-GR samples, respectively, and proportions of HBA and HBB reads to mapped reads were significantly reduced to 5.2% from 26.1% and to 3.7% from 20.1%, respectively (p <0.001, Figure 1). Considering that human globin transcripts constitute 50-70% of the blood RNA [3, 4], the level of pig globin transcripts in pre-GR samples is comparatively low. A possible explanation for the lower level of porcine globin transcripts is that the pigs used in this study were only 1-2 months old, an age associated with rapid decreases in erythrocyte population size and hemoglobin concentration. Although pigs at birth have similar hematological values to adult pigs, by three days of age a 25% reduction in hemoglobin concentration has occurred and hemoglobin concentration then increased gradually from the age of 3 months due to the pig’s tremendous early growth rate, as much as eight times faster than humans [13, 14]. Thus, we expect the GR protocol will reduce more globin transcripts in newborn and adult pig blood RNAs.Table 2


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)

The average proportions ofHBAandHBBreads to total mapped reads in pre- and post-GR samples. Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of HBA and HBB reads to mapped reads significantly (Paired Wilcoxon signed rank test, p <0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: The average proportions ofHBAandHBBreads to total mapped reads in pre- and post-GR samples. Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of HBA and HBB reads to mapped reads significantly (Paired Wilcoxon signed rank test, p <0.001).
Mentions: More than 653 million (M) sequence reads generated from 12 pre- and post-GR samples passed Illumina’s CASAVA (v.1.8) filtration (Table 2). These reads were then aligned to the pig genome build 10.2 by TopHat (v. 2.0.8). After GR treatment, total filtered reads and mapped reads were reduced by an average of 6.1 M and 6.8 M reads, respectively, and globin reads were reduced by an average of 11.4 M reads. The percentage of globin reads among all aligned reads averaged 46.1% and of these, 84.7% were removed by GR treatment. The proportion of globin reads to mapped reads were 46.1% and 8.9% in pre- and post-GR samples, respectively, and proportions of HBA and HBB reads to mapped reads were significantly reduced to 5.2% from 26.1% and to 3.7% from 20.1%, respectively (p <0.001, Figure 1). Considering that human globin transcripts constitute 50-70% of the blood RNA [3, 4], the level of pig globin transcripts in pre-GR samples is comparatively low. A possible explanation for the lower level of porcine globin transcripts is that the pigs used in this study were only 1-2 months old, an age associated with rapid decreases in erythrocyte population size and hemoglobin concentration. Although pigs at birth have similar hematological values to adult pigs, by three days of age a 25% reduction in hemoglobin concentration has occurred and hemoglobin concentration then increased gradually from the age of 3 months due to the pig’s tremendous early growth rate, as much as eight times faster than humans [13, 14]. Thus, we expect the GR protocol will reduce more globin transcripts in newborn and adult pig blood RNAs.Table 2

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