article: Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.
  The contribution of photosynthetic algae to the maintenance of global ecological health is well recognized [1,2]. As primary producers, microalgal species support the survival of organisms at every trophic level. Additionally, they serve as important contributors to the earth’s geochemical cycles. Remarkably, ‘omics’ level characterization of algae is sorely lacking when compared to organisms related to human health or plant biotechnology [3]. Among algae, haptophytes represent an ancient and diverse lineage of eukaryotes. Estimates suggest that photosynthetic members of this taxon were present by the Neoproterozoic era (1000–520 Ma) [4]. The impact of haptophytes on global ecology (i. e. , CO2 sequestration [5] and the production of dimethyl sulfide [6]), toxicity (i. e. , polyketide products [7]); foam and mucilage production [8]; and their trophic value, given their high levels of fatty acids, has long been recognized [9]. However, metagenomic studies have only recently revealed the true extent of haptophyte dominance in aquatic ecosystems [10,11]. Data suggest that these algae may contribute “30 to 40% of the total photosynthetic standing stock in the world’s oceans” [12], and studies show previously unrecognized dominance in fresh water lakes [13]. The fact that some haptophyte species are mixotrophic as well as photosynthetic most likely provides a fitness versatility that helps explain the prevalence of these organisms within algal populations [14]. Two subclasses of haptophytes are recognized [15]—the Pavlovophycidae (single clade) and the Prymnesiophycidae (encompassing 5 clades). Emiliania huxleyi (Isochrysidales) represents the only haptophyte for which a nuclear genome has been published to date [16]. This alga is a marine species well known for its calcified scales [17] and forms large blooms that are visible from space [18]. Genomic studies of the E. huxleyi pan-genome show significant genome variability among strains [19]. The haptophyte characterized in this study is Chrysochromulina tobin (Prymnesiophycidae: B2 clade). Members of the B2 clade to which this alga is associated have been shown to dominate some marine and freshwater ecosystems. For example, data suggest that >55% of all haptophyte sequences in a Mediterranean sampling site and ∼30% in a Norwegian sampling site were of the B2 haptophyte group [11,20,21]. C. tobin (Fig 1) is halotolerant, living in fresh to brackish water [22], and is phagocytotic, using a long haptonema to acquire prey. Unlike many haptophytes that are embellished with either organic or calcium carbonate scales [23], this organism is naturally wall-less. Though small (∼4. 0 μm), 40% of its dry weight is lipid, with most fatty acids stored in two large, well-defined lipid bodies. This study provides insight into several previously unreported genetic characteristics of a haptophyte. The fatty acid production pathways and their potential relationship to changes in lipid body morphology are examined. Furthermore, we identify several novel genes including a polyketide synthase-nonribosomal complex; genes encoding MATE antimicrobial products; the bacterially sourced, laterally transferred genes that encode RuBisCO activase isoforms and a unique xanthorhodopsin. Genomic sequencing and annotation of a representative in the Prymnesiales B2 clade provides critical information to resolve elusive evolutionary relationships among the deeply-rooted haptophyte algal taxon [24,25]. Additionally, genomic knowledge of haptophytes will enhance commercial endeavors that target aquaculture feed stocks, nutraceuticals, plastics or biofuel production. Given that many haptophytes are also toxic, genomic information will support efforts to understand the fundamental metabolic processes associated with the genesis of harmful algal bloom events.
  Chrysochromulina tobin was isolated from a freshwater source, monocultured and bacterial contaminants reduced using reiterative cell sorting by flow cytometry followed by sequential antibiotic treatment. Purified total genomic DNA was used to prepare libraries for 454 and Illumina sequencing. The resulting draft assembly consisted of 3,472 contigs having an average length of ∼17 kb (Table 1). A 59 Mb genome was assembled representing an average read depth of over 100x (see Materials and Methods). The final genome assembly was found to contain a complete genomic sequence of one commensal bacterium (Sphingomonas sp.), that was removed from the assembled genome. Maintenance of C. tobin cultures depends on a light/dark photoperiod (continuous light leads to poor growth). A 12 hour light: 12 hour dark photoperiod partially synchronizes the culture (Fig 2A). Cell division initiates at approximately the tenth hour in the light (L10) and continues at a high rate with the onset of darkness, terminating approximately 6 hours in the dark (D6). Cultures double every 24 hours on a 12 hour light: 12 hour dark cycle in the logarithmic growth phase (Fig 2A). Most notably, during this biphasic 24 hour growth cycle, a dramatic change in the size of the lipid body is observed. The lipid bodies continuously increase in size during the light photoperiod, then shrink rapidly during the dark cycle (Fig 2B). To better understand how light-entrained gene expression controls cell physiology, the entire transcriptome of C. tobin was profiled over the light-dark cycle. RNA was collected at 7 time points during the 24 hour cycle (NCBI: SRX1009273) and gene expression levels were calculated (S1 Dataset: 7 time point expression levels for all genes). Genes with the largest expression changes and large transcript variance between two or more time points were identified and binned into 7 groups for analysis (Fig 3 and S2 Dataset complete list of group members). Group 1 represents genes whose expression peaks midway through the dark period (D6). In this group, transcripts which encode for proteins associated with cell growth and post cell division processes including cytoskeletal changes, and cell projection (microtubular and flagellar components) are overrepresented. Group 2 displays genes that are highly expressed at the end of the dark cycle (time point D10); overrepresented gene categories include genes encoding proteins responsible for metal ion binding, as well as a variety of mitochondrial genes that encode both transporters and ion exchange machinery (e. g. , ferredoxin and rubredoxin). Transcription during this time period also seems to favor the onset of energy molecule and carbohydrate synthesis (Fig 4). Nitrite and phosphate transporters are also observed in this expression cluster, supporting the acquisition of metabolites needed for carbohydrate and fatty acid production. Group 3 genes are highly expressed during D6 and L1. These genes include the following associated gene ontology (GO) terms: ribonucleoside binding (i. e. , purine ribonucleoside binding), GTPase activity, microtubule-based movement, GTP binding, ATP binding, anchoring to plasma membranes, and tubulin. Group 4 gene expression is highest at the beginning of the light period. Thylakoid, chloroplast, photosynthetic, metabolic, glycolytic, and fatty acid biosynthetic processes are prominent in the GO term list. Group 5 includes genes that are highly expressed from L4 to D1. Overrepresented GO terms include genes linked to post transcriptional regulation of gene expression, negative regulation of cellular processes, responses to biotic stimuli, and defense responses. Group 6 genes are upregulated from L1 to L6 and include the GO terms photosystem I and II, chloroplast thylakoid membrane, cell wall, defense response to fungus, response to heat, and ATP binding. Group 7 has significant over representation of ribosomal subunit gene expression (Fig 4). Upregulation of these genes occurs from the middle to the end of the day, with the majority of members showing maximum expression at hour 10 in the light cycle. Though ribosomal sequences represent < 0. 5% of genes annotated, over 38% of genes identified in this group are structural constituents of ribosomes. It is established that ribosomal accumulation usually occurs during the G1 phase of the cell cycle, thereby fostering the production of new biomass ultimately needed to support cell division [32]. Light is a critical factor in the regulation of algal growth [33–35]. As seen above, photoperiod drives a wide range of gene expression rhythms in C. tobin. The predictability of these temporal programs provides several excellent metabolic targets, such as lipases, for genetic engineering [36,37] and may be of special interest to commercial growers who are dependent on seasonal light availability to serve large-scale algal production efforts. The high fatty acid content of algae such as C. tobin can be harnessed as a source for valuable products such as nutraceuticals, alternative energy, or plastics [37,38]. Additionally, the abundant and complex fatty acid composition of haptophytes has historically made these algae highly valued for aquaculture feedstocks and contributed to their broad currency as a food source in aquatic ecosystems [39]. C. tobin produces almost half of its dry weight as lipids [22], much of which is stored in the prominent lipid bodies of each cell. Because lipid body biogenesis is simple (only two lipid bodies) and predictable (regulated by the cell cycle), C. tobin is a viable model organism to study the metabolic processes associated with the genesis of this organelle. Indeed, flow cytometric analysis of C. tobin cells stained with the lipid dye BODIPY 505/515 shows fatty acid content per cell increases during the light and decreases during the dark phases of cell growth (Fig 5). To gain insight into the potential relationship between the observed changes in lipid body size and fatty acid biosynthesis, the regulation of genes important to fatty acid production and loss were inventoried as cells progressed through a single light/dark photoperiod. C. tobin fatty acid biosynthesis pathways are presented in Fig 6 (after [37]). Two major gene expression patterns are notable. The first pattern relates to genes associated with chloroplast-localized fatty acid synthesis, while the second involves transcripts encoding enzymes that convert fatty acids into triacylglycerols (TAGs) after chloroplast export (Fig 6). Transcripts that encode enzymes for converting Acetyl-CoA to Acyl-ACP (e. g. , ACCase, acetyl-CoA carboxylase [Ctob_003321]; KAS, 3-oxoacyl-ACP synthase III [Ctob_004088]; KAR, beta-ketoacyl-ACP reductase [Ctob_008890]; HD, beta-hydroxyacyl-ACP dehydratase [Ctob_006212]; ENR, enoyl-ACP reductase [Ctob_006649]) have increased transcript abundance at the end of the dark photoperiod. This expression pattern also applies to the glycerol-3-phosphate dehydrogenase [Ctob_011737], the enzyme required for readying the glycerol in triacylglycerol (TAG) products. Expression initiates at D10 and peaks at L1 with FPKM (fragments per kilobase of exon per million fragments mapped) [40] peaking at 3 to 500 fold over the lowest value for the aforementioned genes. This expression variability suggests that production of triacylglycerol (TAG) related gene transcripts may be minimal during the dark period. In the second pattern observed, a gradual increase of transcripts encoding enzymes that convert acyl-CoA to TAG products is observed from L4 to L10 (e. g. , GPAT, glycerol 3-phosphate-o-acyltransferase [Ctob_005527]; LPAAT, lysocardiolipin acyltransferase [Ctob_003757]; lysophosphatidic acid phosphatase [Ctob_007879]; LPAT, phospholipid glycerol acyltransferase family protein [Ctob_012317]; DAGAT, diacylglycerol acyltransferase family protein [Ctob_008970]). FPKM value fold change is more modest for these ER-associated transcripts, ranging from 2–4 fold increased transcript abundance during the peak at L10. Interestingly, the last step (DAGAT) of this ER associated metabolic pathway appears to follow the chloroplast gene expression pattern in that transcript expression peaks at L1. Besides differing in the timing of peak transcript abundance and fold-increase, there is a significant difference between the overall transcript levels for the two groups described above. Chloroplast associated processes are generated by very highly transcribed genes with maximum FPKMs ranging from 70 to almost 1500, compared to the ER related processes that have maximum FPKM transcript levels about 8 to 35. These processes may contribute to the rate limiting steps of TAG production, and suggest that ER related reactions may be candidates for overexpression experiments. Fatty acid elongase and desaturase transcripts (S2 Fig) appear to follow the trend of the chloroplast associated gene products, in that genes encoding these enzymes consistently peak in their expression at the L1 time point. However there is great variance among overall transcript levels for each gene queried. In contrast, the gene expression patterns of lipases and esterases parallel those seen in the ER related lipid biosynthesis genes—slowly increasing in the light portion of the photoperiod (S3 and S4 Figs). Similarly, when fluorescein diacetate is used to monitor lipase/esterase levels in synchronized C. tobin cultures, a slow increase in enzymatic activity is seen from L6 to the onset of dark, followed by a rapid decline (Fig 5). This change in enzyme activity correlates with the concentration of lipid found in the cell. Incorporation of the fluorescent dye BODIPY 505/515 shows lipid levels to peak at ∼L9, and then quickly drop as cells enter the dark phase of growth. Taken together, shifts in gene expression, enzymatic activity and lipid concentration suggest that a sequential cascade of metabolic programs (fatty acid synthesis up-regulation in the light, and lipase activity in the dark) drive significant and predictable changes in C. tobin fatty acid storage levels, and that these changes are reflected in the lipid body volume noted above (Fig 2). The data presented here give insight to C. tobin fatty acid biosynthesis. However, as seen in Fig 7, not all haptophytes generate the same quantity or type of fatty acids, even when organisms are maintained under similar physiological conditions. Gas chromatography-mass spectrometry (GC/MS) data presented in Fig 7 and S3 Dataset, comparing fatty acid profiles obtained from a broad sampling of haptophytes clearly demonstrate that even those haptophytes clustered within a specific taxonomic rank may greatly differ in the type and amount of lipids present. Given that biological findings often lead to metabolic engineering efforts, such information potentially facilitates engineering approaches in commercial algal applications. C. tobin has a war chest of defense related genes. The requirement for defense systems is two-fold: first, the alga may need to cope with potentially harmful eco-cohorts, and second, because C. tobin is mixotrophic and actively phagocytotic, ingested prey must be neutralized. A variety of these putative defense mechanisms identified in C. tobin are described below. Many algae have evolved methods that augment survival either by optimizing energy capture [69] or by circumventing the need for synthesizing required compounds [70]. Both of these options appear to be used by C. tobin. Three recovered contigs represent organellar genome sequence and were removed prior to nuclear gene calling and annotation. Read depth of these organelles suggests a copy number of ∼250 chloroplast and ∼800 mitochondrial copies per haploid cell. Complete C. tobin mitochondrial and chloroplast genomes are presented in detail elsewhere [30]. Briefly, the 34,288 kb mitochondrial genome encodes 48 genes. This genome has a large 9. 3 kb repeat section comprised of three large tandem repeats of ~1. 5 kb flanked by smaller repeats—similar to that observed in diatoms [93] and cryptophytes [94]. The 104,518 kb chloroplast genome encodes 145 genes. Similar to rhodophyte, stramenopile and other haptophyte plastids, the C. tobin chloroplast genome contains a preponderance of small, inverted repeats (rather than tandem repeats that dominate in chlorophytic chloroplast genomes) and several unique genes. Chrysochromulina tobin represents the second haptophyte whose genomes (nuclear and organellar) have been sequenced. The nuclear genome is small, compact, gene-rich, and provides evidence of lateral gene transfer events that have contributed to the evolutionary restructuring of this taxon. Transcriptomic data over a 24-hour light: dark cycle reveals a photoperiod-linked gene expression program that is linked to key features of metabolism including lipid biosynthesis and degradation. These data provide the basis for using Chrysochromulina tobin to study lipid body biogenesis. Because C. tobin is phagocytotic, genes encoding host defense were anticipated, and were identified in the form of genes encoding potential antibiotics, antibiotic extrusion proteins, as well as novel antibacterial peptides. C. tobin also represents the first alga for which a polyketide synthase-non ribosomal peptide synthetase (PKS-NRPS) has been identified. This finding may provide potential routes for the synthesis of useful novel metabolites and therapeutics. Chrysochromulina tobin also encodes the first documented xanthorhodopsin in a non-dinoflagellate eukaryote, and led to the identification of equivalent genes in haptophyte, dinoflagellate and cryptophyte species with the cryptophyte rhodopsin-like proteins forming a phylogenetically unique clade that warrants further investigation. Efficient CO2 utilization requires the presence of a support activase. The presence of two RuBisCO activase copies were identified in the C. tobin genome (one nuclear and one chloroplast encoded). This observation was extended to show that all haptophytes, cryptophytes, and stramenopiles for which nuclear and chloroplast genomes are available, have an identical coding profile for these activases. The requirement for exogenous B12 acquisition demonstrates co-dependence of this organism on eco-cohorts for survival. In summary, the C. tobin genome has provided a wealth of new information. Observations reveal products that may be potentially useful in therapeutic application (e. g. , xanthorhodopsins, polyketides) as well as data that may be of high value to algal commercialization and genetic engineering efforts.
  Chrysochromulina tobin strain CCMP291, acquired from The National Center for Marine Algae by the Cattolico laboratory in 2006, was designated as P3. These cultures were maintained in 250 mL Erlenmeyer flasks containing 100 mL of RAC-1, a proprietary fresh water medium. Flasks were plugged with silicone sponge stoppers (Bellco Glass, Vineland, NJ) and capped with a sterilizer bag (Propper Manufacturing, Long Island City, NY). Large volume experimental cultures for genomic DNA and transcriptomic RNA harvesting were maintained in 1. 0 L of RAC-1 medium that was contained in 2. 8 L large-mouth Fernbach flasks. These flasks were plugged with hand-rolled, #50 cheese cloth-covered cotton stoppers and covered with a #2 size Kraft bag (Paper Mart, Orange, CA). All cultures were maintained at 20°C on a 12 hour light: 12 hour dark photoperiod under 100 μEm-2s-1 light intensity using full spectrum T12 fluorescent light bulbs (Philips Electronics, Stamford, CT). No CO2 was provided and cultures were not agitated. Algal cultures were treated in the following manner to minimize bacterial contamination. P3 cultures were subject to re-iterative cell sorting using flow cytometry. C. tobin cells were stained for identification using BODIPY 505/515 (4,4-difluoro-1,3, 5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene; Invitrogen, Carlsbad, CA), a neutral lipid binding fluorophore. Approximately 10 stained cells were sorted into a single well of a 96 well plate containing 100 μL RAC-1 medium and then transferred to 10 mL of RAC-1 medium in 50 mL plastic tissue culture flasks (Nunc, Roskilde, Denmark). This cell sorting process was carried out 4 times with the resulting culture being designated as P4. Cells obtained from reiterative flow cytometric selection (P4) were then treated in RAC-1 medium that contained either streptomycin (resulting in culture P5. 5) or hygromycin (P5. 6). Treatment with these two antibiotics was identical. Cells were exposed to a final concentration of 400 μg/mL antibiotic for 18 hours before 5 mL of treated cultures were transferred to 100 mL of antibiotic free RAC-1 medium. Cultures P5. 5 and P5. 6 were periodically tested for bacterial contamination using liquid LB medium made with RAC-1 medium in replacement of water. Sequencing data and recovery of a cultured bacterial isolate has shown that a single bacterial contaminant is still present in the P5. 5 culture. Total genomic DNA was collected from each of the P5. 5 and P5. 6 cultures using the Qiagen Genomic-tip Maxi DNA extraction protocol (Germantown, MD) with the following changes to the standard protocol. 1. 5 x 108 cells were harvested by centrifugation at 5,378 x g for 20 minutes and resuspended in lysis buffer (20 mM EDTA, pH 8. 0; 10 mM Tris-base, pH 8. 0; 1% Triton X; 500 mM guanidine; 200 mM NaCl). After 1. 0 hour incubation at 37°C, RNase A was added to 200 μg/ml final concentration and the mixture incubated for 30 minutes at 37°C. Following the addition of 600 μL Proteinase K (20 mg/ml) (Sigma-Aldrich) incubation was continued at 50°C for 2. 0 hours, mixing every 30 minutes by swirling. DNA preparation was transferred into a Qiagen DNA binding tip (Maxi size) that was equilibrated using the manufacturer’s instructions, and allowed to pass through the tip by gravity, while maintained at room temperature. The tip was then washed twice using Qiagen buffer QC. Fifteen mL of Buffer QF (at 37°C) was added to the tip to elute the DNA. DNA was precipitated by adding 10. 5 mL of 100% room temperature isopropanol followed by centrifugation at 11,220 x g for 20 min at 4°C. The pellet was washed in 4 mL of 4°C 70% ethanol and centrifuged again using the same conditions. The DNA pellet was air dried for 5 min and resuspended in warmed Qiagen buffer EB (50°C) and incubated at 50°C for 2. 0 hours. DNA solution was quantitated using a spectrophotometer and subsequently transferred to 1. 7 mL Eppendorf tubes and stored at -80°C. The C. tobin genome was sequenced using a combination of Illumina and 454 sequencing. For Illumina, two shotgun libraries (2 X 100 and 1 x 150 base pair) were prepared using standard TruSeq protocols and sequenced on an Illumina HiSeq2000. Using the 454 Titanium platform, shotgun single-end and paired-end (10 kb insert) DNA libraries were prepared generating 4. 7 million reads in total. The 454 single end and paired end data (insert size 8180 +/- 1495 bp) were assembled using Newbler, version 2. 3 (release 091027_1459) (Roche). The sequences generated by the Illumina platform were assembled separately with VELVET, version 1. 0. 13 [95]. Consensus sequences from the VELVET and Newbler assemblies were computationally shredded into 10 kb fragments and were re-assembled with reads from the 454 paired end library using parallel‬ Phrap, version 1. 080812 (High Performance Software, LLC). The final draft genome assembly produced over three thousand contigs. Gene annotation was carried out using the MAKER2 training and annotation pipeline [96]. After masking repeated genomic elements using Repeatmasker [97], genes were modeled by combining several methods in MAKER2: a) aligning C. tobin transcriptomic BLASTn hits as EST evidence; b) aligning to Emiliania huxleyi ESTs using tBLASTx; c) using the assembled RNAseq data for gene prediction with Tophat [98] and Cufflinks [99]; d) aligning all CEGMA (Core eukaryotic genes) [100] genes to the C. tobin contigs using BLASTx; e) Augustus [101] for ab initio models trained on the gene structures of Chlamydomonas reinhardtii; f) SNAP [102] for ab initio models trained on Hidden Markov Models (HMMs) of the predicted genes by cufflinks and Tophat models; g) GenemarkES for ab initio gene models [103]. A total of 16,777 genes were annotated using the above method. Of these, 10,293 were supported by BLAST homology using BLAST2GO and 6,484 are considered novel genes. BLAST2GO [104] was used to attach functional annotation to gene call predictions. First, BLASTp was used to search the non-redundant protein database (nr) with a Blast Expect Value cutoff of 1e-6. Blast2Go Mapping was performed followed by Annotation using E-Value-Hit-Filter: 1e-6, Annotation cutoff of 55 and GO weight of 5. These gene annotations were used in the remainder of the gene analyses unless the manual curation of a gene gave evidence supporting a manual annotation. Twelve 1 L cultures were seeded at a starting density of 50,000 cells/ mL, 66 hours prior to the first harvesting time point (Dark hour 6) using inoculation cultures that were maintained for 7 days at standard conditions (see above). Total RNA was purified using a modified TRIzol preparation: 1. 5 x 108 cells were collected per RNA isolation sample. C. tobin cells were centrifuged at 8,600 x g for 20 minutes in 500 mL polypropylene centrifuge bottles. The supernatant was decanted and 5 mL of TRIZOL reagent (Invitrogen) was added to the cell pellet. Cells were resuspended by pipetting and vortexing for 1 minute. The homogenate was transferred equally into four microcentrifuge tubes. To each tube, 250 μL of chloroform was added. Each tube was shaken by hand vigorously for 15 seconds and subsequently centrifuged for 15 minutes at 12,000 x g at 4°C. The mixing and centrifugation was repeated once. After the second centrifugation, the top aqueous phase was transferred to a new microcentrifuge tube being sure not to disturb the lower phenol/chloroform phase. Ice cold isopropanol (625 μL) was added to each of the 4 tubes containing the aqueous phase and incubated at -20°C overnight. The samples were then centrifuged at 12,000 x g for 10 minutes at 4°C. The supernatant was removed and the pellet washed with 1. 25 mL of 75% ice cold ethanol followed by a 5 minute centrifugation at 7,400 x g. The ethanol wash and centrifugation step was repeated one time. The pellet was dried for 10 minutes and resuspended in 30 μL RNase free water (Qiagen). Four samples were combined into a single tube and treated with RNase free DNase for 90 minutes at 37°C. Samples were then cleaned using a Qiagen RNeasy MinElute clean up protocol as specified by the manufacturer’s instructions. Samples were stored at -80°C Poly-A selection was carried out followed by library preparation using a TruSeq library kit (Illumina). Sequencing was done on the Illumina high-seq and generated 100 bp paired reads. For each time point collected, 15–30 million reads were generated. Reads were trimmed and groomed [105]. Tophat version 1. 5 [98] was used to assemble the sequences using the C. tobin draft genome as a reference. Cufflinks (v2. 1. 1) was used to estimate FPKM (fragments per kilobase of exon per million mapped reads) for each transcript at each time point [106]. To determine which subset of the transcriptomic data to include in the global analysis a high expression and high variance selection method was implemented to determine genes that were 1) highly expressed and 2) had great differences in expression between 2 or more time points. This gene selection was done using an in-house derived formula (“MeanNeighbor”) that takes each time point and compares the expression level (FPKM value) average across adjacent time points. This method was implemented in R using the following function: MeanNeighbor = function (x) {mean (abs (x[2: length (x) ]-x[1: length (x) -1) ]) ) }. The top 1000 genes as scored by MeanNeighbor value were then plotted as a heatmap using a normalization constant so that all values of gene expression FPKM could be represented by a relative level between -3 and +3. First, each individual FPKM value was subtracted from each transcript’s average FPKM value of all 7 time points. The resulting value at each time point was then divided by the standard deviation, giving a relative expression level to be used in the generation of the heatmap. The global heatmap was generated using the R library “pheatmap” [107] using the “ward” clustering method. Fisher’s exact test was used in Blast2GO to determine GO term overrepresentation in each group based on the annotation of GO terms by Blast2GO. The p-value cutoff for this was set at 0. 05. For group 3, over 100 members were obtained so the p-value cutoff was lowered to 1e-7 to generate the graphs used in Fig 4. RNA seq data was also assembled de novo to identify genes that may have not been present or were mis-assembled in the final C. tobin nuclear genome draft. Trinity [108] was used to assemble transcripts, which were used to create an additional BLAST database used in identifying NAD genes, cobalamin synthesis, and polyketide related genes in addition to those found in the nuclear genome draft.
summary: Microalgae are important contributors to global ecological balance, and process nearly half of the world’s carbon each year. Additionally, these organisms are deeply rooted in the earths’ evolutionary history. To better understand why algae are such strong survivors in aquatic environments and to better understand their contribution to global ecology, we sequenced the genome of a microalga that is abundant in both fresh and salt water environments, but poorly represented by current genomic information. We identify protein-coding genes responsible for the synthesis of potential toxins as well as those that produce antibiotics, and describe gene products that enhanced the ability of the alga to use light energy. We observed that a day-night cycle, similar to that found in natural environments, significantly impacts the expression of algal genes whose products are responsible for synthesizing fats—a rich source of nutrition for many other organisms.
title: Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)