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Identifying reference genes with stable expression from high throughput sequence data

ABSTRACT: Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes.

ORGANISM(S): Thalassiosira pseudonana CCMP1335

PROVIDER: GSE40509 | GEO | 2013/01/01

SECONDARY ACCESSION(S): PRJNA174194

REPOSITORIES: GEO

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Project description:Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes. Axenic T. pseudonana CCMP 1335 was grown at 14Â°C under 24 hour light (120 Âµmol photons m-2 s-1) after Dyhrman et al. (2012) in f/2 plus silica chelated media made from surface Sargasso Sea water. Nitrate, silica, vitamins, and trace metals were at f/2 concentrations (Guillard and Ryther 1962), while iron and phosphate were modified across treatments. In brief, triplicate cultures of replete (36 ÂµM PO4, 400 nM Fe), P-limited (0.4 ÂµM PO4, 400 nM Fe), Fe-limited (36 ÂµM PO4, 40 nM Fe), and Co-limited (0.4 ÂµM PO4, 40 nM Fe) treatments were harvested when growth deviated from the replete control. Growth was monitored by cell counts. Biomass was harvested onto 0.2 Âµm filters and flash frozen in liquid nitrogen and total RNA was extracted as described in Dyhrman et al. (2012). Tag-seq sequencing of the transcriptome was performed by Illumina with a polyA selection and NlaIII digestion, resulting in 21 bp sequence reads or tags (Dyhrman et al., 2012). Libraries were of varied sizes as follows: replete (~12 million), P-limited (~13 million), Fe-limited (~23 million), and Co-limited (~26 million). Tags were mapped to gene models (predicted protein coding regions) with a pipeline designed by Genesifter Inc., requiring 100% identity and covering 9759 genes. Tag counts within a gene were pooled and normalized to the size of the library, with resulting data expressed in tags per million (tpm). Genes with normalized tag counts less than 2.5 tpm for each of the four treatments were excluded (Figure S1) , leaving 7380 genes in the analysis.

Project description:Phosphorus (P) is a critical driver of phytoplankton growth and ecosystem structure and function in the ocean. Diatoms are an abundant and widespread functional group of phytoplankton that are responsible for significant amounts of primary production in the ocean, however there has not been a comprehensive study of diatom physiological responses to P deficiency. Here, we coupled deep sequencing of transcript tags and quantitative proteomic analysis from the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. The reads (tags) were mapped to the T. pseudonana genome sequence, confirming expression of 91% of the modeled gene set. A total of 318 genes were differentially regulated with a false discovery rate of p<0.05. A total of 1264 proteins were detected, and of those 136 were differentially expressed with a false discovery rate of p<0.05. Significant changes in the abundance of transcripts and proteins were observed and these changes were coordinated for glycolysis, translation, and multiple biochemical responses to P deficiency. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P (DOP) through increased production of alkaline phosphatase metalloenzymes and a diesterase, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to rapidly respond to variations in environmental P availability. T. pseudonana (Strain 1335 from the Provosoli-Guillard National Center for the Culture of Marine Phytoplankton (CCMP)) was grown in a modified f/2 medium made from Sargasso Sea water. Macronutrients and vitamin B12, biotin, and thiamine solutions were treated with prepared Chelex-100 resin to remove trace metal contaminants followed by trace-metal clean syringe sterilization to yield final nutrient concentrations of 882 μM NaNO3 and 106 μM Na2SiO3, and vitamin concentrations of 75 pM, 400 pM, and 60 nM, respectively. The Fe concentration was also modified from f/2 to 400 nM. All conditions were run in triplicate at 14 ºC, in constant light (120 µmol photons m-2 s-1). Cells were grown with f/2 phosphorus concentrations (P-replete; 36 µM PO4) and with low phosphorus concentrations (P-deficient; 0.4 µM PO4). Growth was monitored daily with cell counts. Duplicate P-replete treatments were pooled and harvested in mid log phase, and triplicate P-deficient treatments were pooled, and also quickly harvested onto 2 µm filters at the onset of P depletion. All RNA samples were snap frozen in liquid nitrogen. Replicate P-deficient cultures were refed to 36 µM phosphate at the onset of P depletion, and subsequently resumed growth. Additional growth studies were performed as described above substituting glycerolphosphate and adenosine monophosphate at 36 µM, for the phosphate in the medium.

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Identifying reference genes with stable expression from high throughput sequence data

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