Limits...
RNA-Seq analysis of seasonal and individual variation in blood transcriptomes of healthy managed bottlenose dolphins

View Article: PubMed Central - PubMed

ABSTRACT

Background: The blood transcriptome can reflect both systemic exposures and pathological changes in other organs of the body because immune cells recirculate through the blood, lymphoid tissues, and affected sites. In human and veterinary medicine, blood transcriptome analysis has been used successfully to identify markers of disease or pathological conditions, but can be confounded by large seasonal changes in expression. In comparison, the use of transcriptomic based analyses in wildlife has been limited. Here we report a longitudinal study of four managed bottlenose dolphins located in Waikoloa, Hawaii, serially sampled (approximately monthly) over the course of 1 year to establish baseline information on the content and variation of the dolphin blood transcriptome.

Results: Illumina based RNA-seq analyses were carried out using both the Ensembl dolphin genome and a de novo blood transcriptome as guides. Overall, the blood transcriptome encompassed a wide array of cellular functions and processes and was relatively stable within and between animals over the course of 1 year. Principal components analysis revealed moderate clustering by sex associated with the variation among global gene expression profiles (PC1, 22 % of variance). Limited seasonal change was observed, with < 2.5 % of genes differentially expressed between winter and summer months (FDR < 0.05). Among the differentially expressed genes, cosinor analysis identified seasonal rhythmicity for the observed changes in blood gene expression, consistent with studies in humans. While the proportion of seasonally variant genes in these dolphins is much smaller than that reported in humans, the majority of those identified in dolphins were also shown to vary with season in humans. Gene co-expression network analysis identified several gene modules with significant correlation to age, sex, or hematological parameters.

Conclusions: This longitudinal analysis of healthy managed dolphins establishes a preliminary baseline for blood transcriptome analysis in this species. Correlations with hematological parameters, distinct from muted seasonal effects, suggest that the otherwise relatively stable blood transcriptome may be a useful indicator of health and exposure. A robust database of gene expression in free-ranging and managed dolphins across seasons with known adverse health conditions or contaminant exposures will be needed to establish predictive gene expression profiles suitable for biomonitoring.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3020-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Globin depletion of dolphin blood RNA. a By real-time PCR, HBA was observed to be significantly depleted by nearly 300-fold (t-test, p < 0.05, n = 5), with little change in other genes (t-test, p > 0.05, n = 5). b Large reductions in HBA and HBB were observed by RNA-seq analysis in the absence of expression decreases in other genes (Cuffdiff, FDR < 0.05, n = 5). Due to extremely high expression values, statistical analyses were not performed on HBA and HBB. c Globin depletion of blood RNA resulted in a 10 % increase of identified genes. Statistical significance is denoted by an asterisk
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5016863&req=5

Fig1: Globin depletion of dolphin blood RNA. a By real-time PCR, HBA was observed to be significantly depleted by nearly 300-fold (t-test, p < 0.05, n = 5), with little change in other genes (t-test, p > 0.05, n = 5). b Large reductions in HBA and HBB were observed by RNA-seq analysis in the absence of expression decreases in other genes (Cuffdiff, FDR < 0.05, n = 5). Due to extremely high expression values, statistical analyses were not performed on HBA and HBB. c Globin depletion of blood RNA resulted in a 10 % increase of identified genes. Statistical significance is denoted by an asterisk

Mentions: As an analysis of a preliminary dolphin blood sample, sequenced at a targeted depth of 15 million reads, revealed that approximately 75 % of reads represented hemoglobin sequences (data not shown), we sought to establish a protocol for globin depletion of dolphin blood to increase the breadth of sequence detection. We modified an Affymetrix globin depletion protocol [12, 14] to yield high quality RNA with greatly depleted levels of hemoglobin transcripts. Recovery of RNA following globin depletion was high (median = 100.4 %) and only a minimal decrease in RIN, from 8.4 ± 0.06 to 7.8 ± 0.09, was observed. It has been reported that RNA recovery is low and variable following globin depletion in human and porcine blood [12, 35] and decreased RINs are also common [12, 36], thus it appears the globin depletion protocol performed exceptionally well in dolphin blood. As measured by quantitative PCR, HBA was significantly reduced by 286.4 ± 1.3 fold (t-test, p < 0.05, n = 5), whereas expression of other selected genes was not significantly changed (t-test, p > 0.05, n = 5) (Fig. 1a). Due to limitations in RNA quantity, only a single sample (Kainalu May) was sequenced at a targeted depth of 28 million reads both pre- and post-globin depletion. In this sample HBA was reduced 99.5 %, HBB 92 %, and HBM 35.8 %. To further investigate the effects of globin depletion in our study, five pairs of globin depleted and non-depleted samples, each collected 1 month apart, were analyzed by RNA-seq. HBA and HBB were reduced by 98.8 and 80.9 % respectively (Fig. 1b). This degree of depletion is similar to that observed using this protocol in porcine samples [12]. HBM exhibited a minor, non-significant decrease of 1.3 fold. In addition, ENSTTRG00000012084, annotated as a novel protein coding gene in the dolphin genome, was expressed at exceptionally high levels in non-depleted samples and was virtually undetected in globin depleted samples (99.9 % reduction). While this gene is termed “novel” in the genome annotation, it is located on a gene scaffold containing only hemoglobin genes (HBA, HBM, HBQ1, and HBZ). Blastx searches of the NCBI nr database identify this gene as HBA (E-value = 4e−37). Likewise ENSTTRG00000009506, annotated as a novel protein coding gene in the Ensembl genome, was reduced by 71.6 % following globin depletion. This gene is located on a scaffold with HBB and HBE and blastx searches identified this gene as HBB (E-value = 4e−98). Despite large reductions in expression, this gene and the annotated HBB were the top two most highly expressed genes in globin depleted samples, indicating that further protocol modifications may improve the reduction of HBB. Overall, globin depletion had little impact on expression of other genes, with only 790 (3.7 %) genes showing significantly different expression between depleted and non-depleted samples (Cuffdiff, FDR < 0.05, n = 5). Among these, 357 exhibited 1.6–13.9 fold higher expression in non-depleted samples, while 433 had 1.6–18.4 fold higher expression in globin depleted samples. As these differences may encompass both biological and technical variation, all further analyses were conducted on the globin depleted samples only.Fig. 1


RNA-Seq analysis of seasonal and individual variation in blood transcriptomes of healthy managed bottlenose dolphins
Globin depletion of dolphin blood RNA. a By real-time PCR, HBA was observed to be significantly depleted by nearly 300-fold (t-test, p < 0.05, n = 5), with little change in other genes (t-test, p > 0.05, n = 5). b Large reductions in HBA and HBB were observed by RNA-seq analysis in the absence of expression decreases in other genes (Cuffdiff, FDR < 0.05, n = 5). Due to extremely high expression values, statistical analyses were not performed on HBA and HBB. c Globin depletion of blood RNA resulted in a 10 % increase of identified genes. Statistical significance is denoted by an asterisk
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016863&req=5

Fig1: Globin depletion of dolphin blood RNA. a By real-time PCR, HBA was observed to be significantly depleted by nearly 300-fold (t-test, p < 0.05, n = 5), with little change in other genes (t-test, p > 0.05, n = 5). b Large reductions in HBA and HBB were observed by RNA-seq analysis in the absence of expression decreases in other genes (Cuffdiff, FDR < 0.05, n = 5). Due to extremely high expression values, statistical analyses were not performed on HBA and HBB. c Globin depletion of blood RNA resulted in a 10 % increase of identified genes. Statistical significance is denoted by an asterisk
Mentions: As an analysis of a preliminary dolphin blood sample, sequenced at a targeted depth of 15 million reads, revealed that approximately 75 % of reads represented hemoglobin sequences (data not shown), we sought to establish a protocol for globin depletion of dolphin blood to increase the breadth of sequence detection. We modified an Affymetrix globin depletion protocol [12, 14] to yield high quality RNA with greatly depleted levels of hemoglobin transcripts. Recovery of RNA following globin depletion was high (median = 100.4 %) and only a minimal decrease in RIN, from 8.4 ± 0.06 to 7.8 ± 0.09, was observed. It has been reported that RNA recovery is low and variable following globin depletion in human and porcine blood [12, 35] and decreased RINs are also common [12, 36], thus it appears the globin depletion protocol performed exceptionally well in dolphin blood. As measured by quantitative PCR, HBA was significantly reduced by 286.4 ± 1.3 fold (t-test, p < 0.05, n = 5), whereas expression of other selected genes was not significantly changed (t-test, p > 0.05, n = 5) (Fig. 1a). Due to limitations in RNA quantity, only a single sample (Kainalu May) was sequenced at a targeted depth of 28 million reads both pre- and post-globin depletion. In this sample HBA was reduced 99.5 %, HBB 92 %, and HBM 35.8 %. To further investigate the effects of globin depletion in our study, five pairs of globin depleted and non-depleted samples, each collected 1 month apart, were analyzed by RNA-seq. HBA and HBB were reduced by 98.8 and 80.9 % respectively (Fig. 1b). This degree of depletion is similar to that observed using this protocol in porcine samples [12]. HBM exhibited a minor, non-significant decrease of 1.3 fold. In addition, ENSTTRG00000012084, annotated as a novel protein coding gene in the dolphin genome, was expressed at exceptionally high levels in non-depleted samples and was virtually undetected in globin depleted samples (99.9 % reduction). While this gene is termed “novel” in the genome annotation, it is located on a gene scaffold containing only hemoglobin genes (HBA, HBM, HBQ1, and HBZ). Blastx searches of the NCBI nr database identify this gene as HBA (E-value = 4e−37). Likewise ENSTTRG00000009506, annotated as a novel protein coding gene in the Ensembl genome, was reduced by 71.6 % following globin depletion. This gene is located on a scaffold with HBB and HBE and blastx searches identified this gene as HBB (E-value = 4e−98). Despite large reductions in expression, this gene and the annotated HBB were the top two most highly expressed genes in globin depleted samples, indicating that further protocol modifications may improve the reduction of HBB. Overall, globin depletion had little impact on expression of other genes, with only 790 (3.7 %) genes showing significantly different expression between depleted and non-depleted samples (Cuffdiff, FDR < 0.05, n = 5). Among these, 357 exhibited 1.6–13.9 fold higher expression in non-depleted samples, while 433 had 1.6–18.4 fold higher expression in globin depleted samples. As these differences may encompass both biological and technical variation, all further analyses were conducted on the globin depleted samples only.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: The blood transcriptome can reflect both systemic exposures and pathological changes in other organs of the body because immune cells recirculate through the blood, lymphoid tissues, and affected sites. In human and veterinary medicine, blood transcriptome analysis has been used successfully to identify markers of disease or pathological conditions, but can be confounded by large seasonal changes in expression. In comparison, the use of transcriptomic based analyses in wildlife has been limited. Here we report a longitudinal study of four managed bottlenose dolphins located in Waikoloa, Hawaii, serially sampled (approximately monthly) over the course of 1&nbsp;year to establish baseline information on the content and variation of the dolphin blood transcriptome.

Results: Illumina based RNA-seq analyses were carried out using both the Ensembl dolphin genome and a de novo blood transcriptome as guides. Overall, the blood transcriptome encompassed a wide array of cellular functions and processes and was relatively stable within and between animals over the course of 1&nbsp;year. Principal components analysis revealed moderate clustering by sex associated with the variation among global gene expression profiles (PC1, 22&nbsp;% of variance). Limited seasonal change was observed, with&thinsp;&lt;&thinsp;2.5&nbsp;% of genes differentially expressed between winter and summer months (FDR&thinsp;&lt;&thinsp;0.05). Among the differentially expressed genes, cosinor analysis identified seasonal rhythmicity for the observed changes in blood gene expression, consistent with studies in humans. While the proportion of seasonally variant genes in these dolphins is much smaller than that reported in humans, the majority of those identified in dolphins were also shown to vary with season in humans. Gene co-expression network analysis identified several gene modules with significant correlation to age, sex, or hematological parameters.

Conclusions: This longitudinal analysis of healthy managed dolphins establishes a preliminary baseline for blood transcriptome analysis in this species. Correlations with hematological parameters, distinct from muted seasonal effects, suggest that the otherwise relatively stable blood transcriptome may be a useful indicator of health and exposure. A robust database of gene expression in free-ranging and managed dolphins across seasons with known adverse health conditions or contaminant exposures will be needed to establish predictive gene expression profiles suitable for biomonitoring.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3020-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus