Transcriptomic Basis of Metamorphic Competence in the Salt-Marsh-Dwelling Polychaete Capitella teleta.
Many marine invertebrate species from a wide variety of animal phyla depend on their planktonic larvae for dispersal. These microscopic larvae often spend hours to weeks dispersing in the water column before finally settling to the benthos and initiating metamorphosis (Thorson, 1950; Pechenik, 1990). These larvae finally end their planktonic journey once they perceive a settlement cue--a stimulus from the environment that triggers larvae to settle to the benthos and initiate metamorphosis (Stanley et al., 2010; Hadfield, 2011; Lillis et al., 2013). However, the larvae of many species must develop for hours, days, or weeks before becoming responsive to such environmental triggers (Pechenik, 1990; Hadfield et al., 2001).
Time to metamorphic competence thus determines the minimum amount of time that larvae will spend dispersing in the water column--and the distance they will be dispersed--before they are able to perceive a settlement cue, settle, and initiate metamorphosis. While larvae are progressing toward competence in the water column, they are also developing juvenile structures and organs that are essential for life after metamorphosis; by the time larvae reach competence, they are essentially fully formed juveniles waiting to shed larval structures (Hadfield et al., 2001). While some natural settlement cues have been characterized (Pawlik and Faulkner. 1986; Tebben et al., 2011; Swanson et al., 2012; Burns et al., 2014; Shikuma et al., 2014) and various aspects of the physiological control of metamorphosis have been determined (Bishop and Brandhorst, 2001; Leise et al., 2001; Pechenik et al., 2002, 2007; Bishop et al., 2008; Biggers et al., 2012), almost nothing is known about the molecular or physiological mechanisms controlling the onset of metamorphic competence--one of the key processes that determine potential distributions of marine invertebrate species.
To begin understanding the shifts in gene expression that lead to metamorphic competence in marine invertebrates, we have studied the deposit-feeding salt-marsh polychaete Capitella teleta (Blake. Grassle & Eckelbarger, 2009). Capitella teleta is a model organism with a sequenced and annotated genome that has been used for studying developmental biology, larval settlement, and aspects of the control of metamorphosis (Blake et al., 2009). Larvae of C. teleta will rapidly settle and metamorphose once they contact salt-marsh sediment, often in less than 30 min (Dubilier, 1988; Cohen and Pechenik, 1999). While the larvae of many different species become competent after days to weeks of development in the plankton and at widely different times and sizes within each brood (e.g., Pechenik and Heyman, 1987), the larvae of C. teleta are competent to metamorphose within minutes of release from their brooding structure, a brood tube (Dubilier, 1988; Cohen and Pechenik. 1999).
Mated females of C. teleta soon produce a brood tube out of mucus and sand grains and deposit their embryos on the inner walls of the tube (Blake et al., 2009). Embryos develop about 8 d before they escape and swim as larvae into the water column (Seaver et al., 2005). While most larvae that are released from brood tubes are immediately competent to metamorphose, a brood is occasionally released with larvae that are still precompetent (W. J. Biggers, R. Burns, and J. Pechenik, pers. obs.). These advanced "stage 8" precompetent larvae of C. teleta have a conspicuously straight gut morphology, while the "stage 9" competent larvae have a distinctive spiral gut morphology (Seaver et al., 2005). No more than 24 h of development separate these 2 stages (Seaver et al., 2005). Thus, C. teleta is especially well suited to explore the shifts in gene expression that cause larvae to become competent: larvae become competent within 30 min of their release into the plankton, and the onset of competence is essentially synchronous within a brood. Once released from the brood tube, the planktonic larvae locomote within the water column using their ciliary trochal bands. On reception of a settlement cue, the larvae then settle to the substrate and initiate metamorphosis. During metamorphosis, the once-planktonic larva's body elongates, trochal cilia are shed, the prostomium elongates, and capillary setae protrude (Biggers et al., 2012). Newly metamorphosed juveniles then begin burrowing and eating sediment.
Here, we took advantage of the already assembled and annotated C. teleta genome and easily identifiable precompetent and competent larvae to conduct Illumina mRNA sequencing of these two larval stages, enabling us to identify patterns of differential gene expression at the onset of competence. While we expected some shifts in gene expression to be associated with the onset of metamorphic competence, other shifts may anticipate the individual's needs during or immediately after metamorphosis. By understanding how larvae become competent to metamorphose, we will piece more of the signal transduction cascade that regulates settlement and metamorphosis together. Which genes must be expressed at higher levels in competent larvae to allow signal detection and metamorphosis to occur? These gene products could be present in higher concentrations in competent larvae but be missing or in lower concentrations in precompetent individuals. Precompetent larvae could also be producing inhibitory substances in higher concentrations than larvae that have attained competence (Bishop and Brandhorst, 2001; Pechenik et al., 2007; Biggers et al., 2012). Understanding these transcriptomic changes between two stages that are so very close to each other in developmental time yet functionally very different will aid our understanding of what enables metamorphosis to take place.
Materials and Methods
Animal culture and collection of larvae
Starter cultures of adult Capitella teleta were reared in 12.7-cm-diameter glass dishes containing 30 psu of Instant Ocean artificial seawater (ASW) and maintained at 16 [degrees]C. The adults were fed sediment collected from the Little Sippewissett Salt Marsh (Falmouth. MA). Salt-marsh sediment was first passed through a 1-mm sieve and frozen at -20[degrees]C for at least 24 h before being used as food or in settlement experiments. Culture dishes were searched regularly for brood tubes, which were then transferred individually to 5-cm-diameter glass dishes containing only ASW. Sand grains were carefully cleared from the side of the brood tubes to allow us to observe the developing embryos. This did not disturb the developing embryos or the brooding mother, as the mucous tube below the sand grains remained intact. Once embryos developed to stage 8 (straight gut) within the brood tube, the tube was carefully cut open; the stage 8 larvae were allowed to swim out and were collected for tissue and experiments. Stage 9 larvae (spiral gut) were collected for tissue preservation and experiments when they naturally escaped from the brood tube. All larval observations were made at 12-50 x magnification using a dissecting microscope.
Testing larvae for metamorphic competence
To determine whether stage 8 larvae were indeed precompetent and stage 9 larvae were competent to metamorphose, we placed the larvae in ASW containing salt-marsh sediment and monitored them for metamorphosis. Three replicates of 10 larvae per stage were added to 4 ml of ASW in plastic 16-well plates with each well containing 1.72 [cm.sup.3] of saltmarsh sediment. Wells were observed for metamorphosed juveniles every 30 min for 5 h at 50 x using a dissecting microscope, with a final observation at 24 h.
Illumina mRNA sequencing library preparation
Stage 8 and 9 larvae were preserved separately after being released from their brood tubes in RNAlater solution (Thermo Fisher Scientific, Waltham, MA) and stored at -80 [degrees]C. Larval mRNA was later extracted by grinding the larvae in 1.5-ml microfuge tubes with a plastic pestle and then purified using Qiagen RNeasy Mini Kits (Qiagen, Hilden, Germany) with a final elution volume of 20 [micro]l. We used a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) to determine that 50 larvae needed to be pooled in one extraction to yield the 1 [micro]g of mRNA required by the Illumina TruSeq mRNA Library Preparation Kit (ver. 2.0) (Illumina, San Diego, CA). Four replicate larval mRNA extractions were made for each larval stage, and each replicate was made into a uniquely barcoded individual mRNA sequencing library using the Illumina TruSeq mRNA Library Preparation Kit (ver. 2.0). The eight individual libraries were then sent to the Tufts University Core Facility, where they were pooled at equal concentrations and sequenced in one Illumina HiSeq 2500 lane to yield 50-bp-long single-ended reads. All libraries were uploaded to the National Center for Biotechnology Information (NCBI) Short Read Archive and are associated with NCBI BioProject PRJNA379706.
Sequence quality control, alignment, and differential expression analysis
Each library was assessed for sequence quality and potential adapter or primer contamination using FastQC software (Babraham Bioinformatics, 2017). All reads were processed with Trimmomatic software (ver. 0.35) in single-end mode to remove contaminating Illumina sequencing adapters and low-quality sequences (Bolger et al., 2014). Here, starting and ending bases of a read with a Phred score less than 3 were trimmed away. After this, a sliding window was applied to cut low-quality sequences away from a read if the average base quality within a 4-bp window fell below a Phred score of 15. Only reads that were at least 36 bp long were retained for alignment. Each trimmed library was then aligned to the C. teleta genome obtained from the Ensembl Metazoa database (ver. 82.1) (International Nucleotide Sequence Database Collaboration assembly GCA_000328365.1) (Simakov et al., 2014; Kersey et al., 2016), using the default settings of Bowtie 2 (ver. 2.2.5) (Langmead and Salzberg, 2012) and TopHat 2 (ver. 2.0.14) (Trapnell et al., 2009) software. The percentage of bases aligning to different genomic features, such as coding regions, untranslated regions (UTRs), intronic regions, intergenic regions, and rRNA genes, was determined using the Picard tool Collect-RnaSeqMetrics (ver. 2.9.0) (Broad Institute, 2017). HTSEq-count software was used to determine the number of reads that aligned to each gene within each tissue library replicate (Anders et al., 2015). The R package EdgeR was then used to normalize libraries by scaling for library size, estimate dispersions by the quantile-adjusted conditional maximum likelihood method, and calculate differential expression using quasi-likelihood F-tests, all of which are recommended for RNA sequencing experiments with a single factor (Robinson and Smyth, 2007a, b; Robinson et al., 2009; Robinson and Oshlack, 2010; Lund et al., 2012; McCarthy et al., 2012; Chen et al., 2014).
Gene ontology analysis
Genes were considered differentially expressed if they had at least a twofold increase or decrease in expression level between developmental stages, a P-value of <0.05, and a false discovery rate (FDR) of <0.05. Gene ontology classifications for annotated genes were obtained from the Ensembl Metazoa BioMart C. teleta database (ver. 1.0) (Kinsella et al., 2011). A Fisher's exact test was conducted in Blast2GO software to identify significantly (FDR < 0.05) enriched and depleted gene ontology categories between precompetent and competent larvae (Conesa et al., 2005).
Tests of metamorphic competence
All stage 9 larvae metamorphosed and burrowed within 5 h of contact with salt-marsh sediment, while no stage 8 larvae metamorphosed within this time frame (Fig. 1). However, all larvae from both developmental stages had metamorphosed within 24 h after the start of the treatment.
Sequencing and alignment results
A total of 200,064,476 50-bp reads were successfully demultiplexed into 8 separate libraries from the single Illumina HiSeq 2500 sequencing lane. The mean base-call quality of all bases in the demultiplexed reads surpassed a Phred quality of 30 (less than 1/1000 chance a base was called incorrectly). After quality and adapter trimming, the mean library size was 24,677,106 reads; the smallest library contained 19,434,838 reads, while the largest library contained 28,965,035 reads (Table 1). At least 94.9% of reads per trimmed library were successfully aligned to the Capitella teleta genome. The majority of bases that aligned for each library had aligned to genomic regions contributing to mRNA (coding regions and UTRs); an average of 74.0% of bases had aligned to these regions. Ribosomal RNA contamination does not appear to have severely affected library construction; a maximum of 3.9% of bases had aligned to rRNA genes (Table 1).
A total of 2605 genes were significantly differentially expressed between the stage 8 precompetent larvae and the stage 9 competent larvae (Fig. A1). Of these, 1536 genes were expressed at higher levels in the precompetent larvae, while 1069 genes were expressed at higher levels in the competent larvae (Table S1, available online). Qualitative multidimensional scaling analysis of all samples revealed that replicates of each larval stage mainly cluster together and that one dimension separates the replicates of each larval stage (Fig. 2).
This indicates that gene expression patterns are more similar for replicates within each larval stage and are more different between larval stages. Of the 2605 differentially expressed genes, 796 were not annotated with a gene ontology in the Ensembl Metazoa BioMart C. teleta database (ver. 1.0).
Enriched gene ontologies for each larval stage
Overall, it appears that precompetent larvae are continuing larval morphological and physiological development while also increasing the expression of genes coding for proteins that may inhibit metamorphosis (Table 2). Here, genes involved in processes like protein folding, protein transport, translation initiation, and DNA replication are expressed more highly in precompetent larvae. In contrast, competent larvae are mainly increasing the expression of genes with potential chemosensory functions as well as neurotransmitter receptors that could be involved in the signal transduction cascade leading to metamorphosis (Table 3).
Downregulated genes in competent larvae
A fair number of genes were more highly expressed in precompetent larvae than in metamorphically competent larvae.
Most of the gene ontologies that were more highly expressed by precompetent larvae are associated with growth and development. These gene ontologies include categories involved with DNA replication, cell division, transcription, translation, protein folding, and other protein modifications (Table 2). These results agree with Hadfield et al.'s (2001) suggestion that all juvenile structures required for postmetamorphosis juvenile life must be developed before a larva attains metamorphic competence. Through metamorphosis larval-specific structures are shed, and then, because juvenile features required for benthic life are already developed, individuals can immediately proceed further with growth and development into adults. Indeed, gut development is not completed in precompetent stage 8 larvae of Capitella teleta, and circular, longitudinal, and oblique muscles are not well developed prior to the stage 9 competent larvae of C. teleta (Seaver et al., 2005); these features are required for successful burrowing and deposit feeding in C. teleta juveniles. In addition, embryos and larvae of C. teleta continue producing new segments until they reach metamorphic competence at stage 9, with released larvae halting further segmentation until metamorphosing with a total of 12-14 segments (Seaver et al., 2005). Even development of the animal's nervous system halts once the larva reaches competent stage 9. Surprisingly, the development of C. teleta's nervous system could be considered complete once larvae reach stage 9, as the nervous system changes little between stage 9 larvae and the newly metamorphosed juveniles (Meyer et al., 2015).
Some genes responsible for the synthesis of nitric oxide and other processes that could inhibit metamorphosis were also more highly expressed in the precompetent larvae of C. teleta. The competent larvae of C. teleta, along with those of some molluscs, ascidians, and echinoderms, are inhibited from metamorphosing by endogenously produced nitric oxide (Bishop and Brandhorst, 2001; Pechenik et al., 2007; Biggers et al., 2012; for examples of nitric oxide stimulation of metamorphosis in an ascidian and an abalone. see Ueda and Degnan, 2013, 2014). Not surprisingly, in many animals nitric oxide synthase (NOS) requires an interaction with heat-shock protein 90 (HSP90) to keep the essential heme cofactor bound (Bishop and Brandhorst, 2003). However, some competent hydroid and gastropod larvae metamorphosed after being stressed with increased temperatures (Kroiher et al., 1992; Gaudette et al., 2001; Bishop and Brandhorst, 2003), probably because HSP90 proteins were diverted from NOS to renature proteins that had denatured from thermal stress (Bishop and Brandhorst, 2003). Both HSP70 and HSP90 were more highly expressed in precompetent larvae of C. teleta and were downregulated in competent larvae (Table A1). While these proteins also play roles in developmental processes, it is possible that increased expression of HSP90 could ensure sufficiently high endogenous concentrations of nitric oxide to prevent metamorphosis from occurring.
Four heme peroxidases were also more highly expressed in precompetent larvae and downregulated in competent larvae (Table A2). Heme peroxidases are known to play roles in innate immunity, hormone synthesis, and prevention of oxidative stress in the cell. Heme peroxidases work by using hydrogen peroxide to oxidize various substrates to produce an oxidized product and water. Hydrogen peroxide has stimulated at least partial metamorphosis in larvae from a number of marine invertebrates (Pires and Hadfield, 1991; Boettcher and Targett. 1998). Two superoxide dismutases, which produce either oxygen or hydrogen peroxide from the reactive oxygen species superoxide, were also more highly expressed in precompetent larvae of C. teleta (Table A2). Precompetent larvae of C. teleta may be inactivating the reactive oxygen species hydrogen peroxide by catalyzing its breakdown into water. Reactive oxygen species such as hydrogen peroxide can inhibit endogenous nitric oxide production (Murad, 2006), and such inhibition of the inhibitory endogenous nitric oxide in marine invertebrates could lead to metamorphosis.
Upregulated genes in competent larvae
The most significantly enriched gene ontology categories within the competent larvae of C. teleta have to do with the gene ontology processes and functions of sodium ion channels and sodium transport. Within these two ontologies, 20 genes have been annotated as being amiloride-sensitive sodium channels (Table A3). In addition, five other genes that were upregulated in competent larvae of C. teleta have been annotated as having similarities to polycystic kidney disease (PKD) proteins (Table A4). Both amiloride-sensitive sodium channels and PKD channels belong to a large family of channels called the degenerin/epithelial sodium channel family (DEG/ENaC family) (Ben-Shahar, 2011). These DEG/ENaC channels perform a variety of sensory functions in vertebrates and invertebrates, including chemo- and mechanosensation. These channels are involved in the reception of salt, sour, and "water" tastes in a variety of different organisms, from humans to the fruit fly Drosophila melanogaster. Mechanosensory functions of these proteins range from skin pressure reception to pain response from applied acids (Ben-Shahar, 2011). Mammalian genomes encode for only eight or nine of these proteins, while the genomes of such invertebrates as D. melanogaster and the nematode Caenorhaditis elegans encode for 30 or 31, making them one of the largest protein families represented among invertebrate genomes; these proteins may have evolved to perform many specialized sensory functions in invertebrates (BenShahar, 2011). Amiloride appeared to be weakly inductive to larvae of the hydroid Hydractinia echinata: a 600 [micro]mol [l.sup.-1] solution of amiloride in seawater induced ~30% of the tested larvae to metamorphose (Berking, 1988). The role that DEG/ENaC channels play in settlement and metamorphosis has not been explored in any other marine invertebrate species. By expressing these channels at higher levels at competence, larvae of C. teleta could be preparing to navigate and assess complex pelagic and benthic landscapes as they prepare for metamorphosis.
The greatest number of genes that were upregulated in competent larvae are found within the membrane cellular component ontology (Table 3). This category contains the sodium channels described above as well as other relevant receptors. The most abundant of these receptors were the 44 G-protein-coupled receptors (GPCRs) (Table A5). GPCRs are a large family of transmembrane chemosensory receptors that are commonly involved in chemical sensation, including taste and smell. This result is similar to GPCR transcription patterns that have been reported for the demosponge Amphimedon queenslandica, in which many GPCRs were well expressed in pelagic larvae and then downregulated in benthic juveniles (Conaco et al., 2012). Here, Conaco et al. (2012) argue that GPCRs may be used by larvae to sense appropriate settlement cues and environments for settlement and metamorphosis (Conaco et al., 2012). GPCRs have often been studied as putative receptors for settlement cues in marine invertebrate larvae (Hadfield, 2011). While GPCRs do not mediate settlement and metamorphosis in larvae of the polychaete Hydriodes elegans or the coral Montipora capitate (Holm et al., 1998; Tran and Hadfield, 2012), metamorphosis of the larvae of the marine mollusc Haliotis rufescens (abalone) did appear to be modulated by a GPCR (Baxter and Morse, 1987). Among the GPCRs expressed in C. teleta, 39 were annotated as rhodopsin-like GPCRs, including a 5-hydroxytryptamine 6 (5-[HT.sub.6]) receptor, a glycoprotein hormone receptor, a thyrotropin-releasing hormone receptor, and a galanin receptor. The upregulation of a galanin receptor in competent larvae of C. teleta is intriguing, as the function of the short neuropeptide galanin in invertebrates is not well understood (Liu et al., 2010). In mammalian species, galanin is involved in regulating food intake, pain reception, and nerve regeneration (Branchek et al., 2000). Interestingly, mammalian genomes (humans, mice, and rats) also encode three nicotinic acid receptors in the A11 subfamily of rhodopsin-like GPCRs (genes HCAR1, HCAR2, and HCAR3). Nicotinic acid and the related B vitamin nicotinamide rapidly stimulated larvae of C. teleta to metamorphose (Burns et al., 2014). However, when BLASTP searches using these human protein sequences were conducted within the C. teleta proteome, no high-quality matches were found (lowest E-value = 3.5E- 14; highest percent identity = 32.6%). Perhaps the ligand-binding channel that larvae of C. teleta use to sense nicotinamide and nicotinic acid is not a GPCR (Bums et al., 2014).
The upregulation of the 5-[HT.sub.6], serotonin receptor in competent larvae of C. teleta is exciting because treating these larvae with serotonin or the selective serotonin reuptake inhibitor fluoxetine stimulates them to metamorphose (Biggers et ai. 2012). In addition, the serotonin receptor antagonist ketanserin is known to inhibit larvae from metamorphosing in response to NOS inhibitors and nicotinamide (Biggers et al., 2012; Bums et al., 2014). Larvae of C. teleta may require serotonin to transduce signals stimulated by the reception of a settlement cue leading to the initiation of metamorphosis, something that could be explored in future studies.
Other GPCRs included five [gamma]-aminobutyric acid (GABA) B receptors. While not a GPCR, a GABA A receptor was also upregulated (Table A6). GABA is an inhibitory amino acid neurotransmitter that has stimulated some species of marine invertebrate larvae to metamorphose (Morse et al., 1979; Pearce and Scheibling, 1990; Garcia-Lavandeira et al., 2005; Yang et al., 2015). Like serotonin, perhaps a component of the signal transduction cascade leading to metamorphosis in C. teleta is mediated by GABA. Other upregulated receptors in competent larvae included seven ionotropic glutamate receptors, two nicotinic acetylcholine-gated receptors, two neurotransmitter-gated ion channels, and a gustatory receptor (Table A6). Although it is not clear what substance this gustatory receptor is responsible for sensing, this is more evidence that competent larvae of C. teleta have upregulated another gene responsible for environmental chemosensation.
Overall, it appears that precompetent larvae of C. teleta are completing the development of structures required for survival as deposit-feeding juveniles and exhibiting increased expression of genes belonging to gene ontologies that reflect this status of continued growth and development (Table 2). However, once this development is completed, competent larvae of this species appear to then downregulate genes related to overall development and growth and subsequently increase the expression of ligand-binding transmembrane channels and other potential chemosensory proteins. Although we do not know to which ligands these proteins bind, the potential for chemosensation is greater in competent larvae because of the increased expression of these genes.
In combination with a potential for greater chemosensation, competent larvae also showed increased expression of many neurotransmitter receptors. Here, serotonin, GABA, glutamate, acetylcholine, and other neurotransmitters may play a role in transducing the signal perceived from a settlement cue to initiate settlement and metamorphosis. Perhaps chemosensory receptors and other components of the signal transduction cascade leading to metamorphosis are highly expressed at competence as a safety mechanism: if individuals of C. teleta were to metamorphose before their muscles, gut. or nervous system were fully functional, the resulting juvenile would not be likely to survive in the benthos. Future studies should examine the role that GPCRs play in modulating settlement and metamorphosis in C. teleta, as so many of these receptors were expressed at higher levels in competent larvae. In addition. the potential for amiloride-sensitive sodium channels to act as chemosensory channels should be explored in the larvae of C. teleta and other marine invertebrates because of their substantially higher expression level in competent larvae of C. teleta. To our knowledge, the possibility that these channels play roles in the control of metamorphosis in the larvae of marine invertebrates has not been thoroughly explored.
We thank Dr. Judith Grassle (Rutgers University) for sending us a population of Capitella teleta to begin our cultures at Tufts University. This work was funded by a Tufts Institute of the Environment fellowship.
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Table A1 Heat-shock proteins upregulated in precompetent larvae of Capitella teleta Gene ID (a) [Log.sub.2] FC (b) [Log.sub.2] CPM (c) F (d) CaptcG18512 2.91 9.25 124.33 CapteG179511 2.60 6.43 84.46 CapteG159587 2.45 9.12 176.43 CapteG221226 2.33 6.96 134.77 CapteG223688 1.96 2.99 23.11 CapteG223831 1.51 6.74 130.54 Gene ID (a) P-value (e) FDR (f) InterPro descriptors (g) CaptcG18512 1.39E-07 3.74E-05 Heat-shock protein 70 family CapteG179511 1.07E-06 1.20E-04 Heat-shock protein 70 family CapteG159587 2.06E-08 1.45E-0.5 Heat-shock protein HSP90 family CapteG221226 8.98E-08 2.95E-05 Heat-shock protein 70 family CapteG223688 4.60E-04 8.42E-03 Heat-shock protein 70 family CapteG223831 1.07E-07 3.20E-05 Heat-shock protein HSP90 family (a) Ensembl Metazoa gene ID. (b) [Log.sub.2] fold change (FC) in gene expression between precompetent and competent larvae of C. teleta. Positive values are expressed more highly in precompetent larvae, while negative values arc expressed more highly in competent larvae. (c) Average [Log.sub.2] read count per gene per million reads mapped (CPM). (d) Differential expression test F-statistic. (e) Differential expression test P-value. (f) Benjamini-Hochberg-adjusted P-value to control type 1 error. FDR. false discovery rate. (g) InterPro annotation descriptions for each gene. Table A2 Heme peroxidases and superoxide dismutases upregulated in precompetent larvae of Capitella teleta Gene ID (a) [Log.sub.2] FC (2) [Log.sub.2] CPM (c) F (d) CapteG134931 4.08 3.93 106.04 CapteG72445 2.67 2.57 44.57 CapteG146425 2.30 1.88 29.96 CapteG191097 1.48 3.54 27.15 CapteG229194 1.12 5.32 41.42 CapteG148692 1.12 3.22 23.22 Gene ID (a) P-value (e) FDR (f) InterPro descriptors (g) CapteG134931 3.25E-07 5.60E-05 Heme peroxidase CapteG72445 2.58E-05 1.04E-03 Heme peroxidase CapteG146425 1.56E-04 3.83E-03 Heme peroxidase CapteG191097 2.37E-04 5.20E-03 Heme peroxidase CapteG229194 3.63E-05 1.35E-03 Manganese/iron superoxide dismutase CapteG148692 4.51E-04 8.29E-03 Superoxide dismutase (Cu/Zn)/ superoxide dismutase copper chaperone (a) Ensembl Metazoa gene ID. (b) [Log.sub.2] fold change (FC) in gene expression between precompetcnt and competent larvae of C. telela. Positive values are expressed more highly in precompetent larvae, while negative values are expressed more highly in competent larvae. (c) Average [log.sub.2] read count per gene per million reads mapped (CPM). (d) Differential expression test F-statistic. (e) Differential expression test P-value. (f) Benjamini-Hochberg-adjusted P-value to control type I error. FDR. false discovery rate. (g) InterPro annotation descriptions for each gene. Table A3 Amiloride-sensitive ion channels upregulated in competent larvae of Capitella teleta Gene ID (a) [Log.sub.2] FC (b) [Log.sub.2] CPM (c) F (d) CapteG204744 -1.09 3.04 14.92 CapteG200711 -1.10 3.36 16.35 CapteG228582 -1.11 3.73 15.12 CapteG146895 -1.21 2.85 12.12 CapteG187791 -1.25 0.11 14.18 CapteG135400 -1.26 1.31 18.74 CapteG191757 -1.28 -0.06 11.58 CapteG63286 -1.41 0.76 16.02 CapteG227962 -1.42 0.93 28.81 CapteG218320 -1.45 1.35 17.83 CapteG207658 -1.48 -0.33 11.73 CapteG188964 -1.56 1.62 28.56 CapteG195930 -1.68 0.82 38.26 CapteG212550 -1.68 0.66 29.43 CapteG73713 -1.71 0.69 15.52 CapteG192207 -1.73 0.44 17.10 CapteG195931 -1.75 0.85 24.02 CapteG215630 -1.79 2.26 23.38 CapteG220280 -1.82 0.93 27.56 CapteG201141 -2.25 3.79 88.46 Gene ID (a) P-value (e) FDR (f) CapteG204744 2.36E-03 2.76E-02 CapteG200711 1.71E-03 2.18E-02 CapteG228582 2.26E-03 2.67E-02 CapteG146895 4.70E-03 4.52E-02 CapteG187791 4.35E-03 4.27E-02 CapteG135400 1.04E-03 1.54E-02 CapteG191757 5.43E-03 4.99E-02 CapteG63286 1.84E-03 2.30E-02 CapteG227962 2.47E-04 5.35E-03 CapteG218320 1.25E-03 1.75E-02 CapteG207658 5.20E-03 4.85E-02 CapteG188964 I.9IE-04 4.44E-03 CapteG195930 5.24E-05 1.73E-03 CapteG212550 1.68E-04 4.03E-03 CapteG73713 2.06E-03 2.50E-02 CapteG192207 1.46E-03 1.95E-02 CapteG195931 3.93E-04 7.50E-03 CapteG215630 4.39E-04 8.13E-03 CapteG220280 2.22E-04 4.98E-03 CapteG201141 8.44E-07 1.04E-04 Gene ID (a) InterPro descriptors (g) CapteG204744 [Na.sup.+] channel, amiloride sensitive CapteG200711 [Na.sup.+] channel, amiloride sensitive CapteG228582 [Na.sup.+] channel, amiloride sensitive CapteG146895 [Na.sup.+] channel, amiloride sensitive CapteG187791 [Na.sup.+] channel, amiloride sensitive CapteG135400 [Na.sup.+] channel, amiloride sensitive CapteG191757 [Na.sup.+] channel, amiloride sensitive CapteG63286 [Na.sup.+] channel, amiloride sensitive CapteG227962 [Na.sup.+] channel, amiloride sensitive CapteG218320 [Na.sup.+] channel, amiloride sensitive CapteG207658 [Na.sup.+] channel, amiloride sensitive CapteG188964 [Na.sup.+] channel, amiloride sensitive CapteG195930 [Na.sup.+] channel, amiloride sensitive CapteG212550 [Na.sup.+] channel, amiloride sensitive CapteG73713 [Na.sup.+] channel, amiloride sensitive CapteG192207 [Na.sup.+] channel, amiloride sensitive CapteG195931 [Na.sup.+] channel, amiloride sensitive CapteG215630 [Na.sup.+] channel, amiloride sensitive CapteG220280 [Na.sup.+] channel, amiloride sensitive CapteG201141 [Na.sup.+] channel, amiloride sensitive (a) Ensembl Metazoa gene ID. (b) [Log.sub.2] fold change (FC) in gene expression between precompetent and competent larvae of C. teleta. Positive values are expressed more highly in precompetent larvae, while negative values are expressed more highly in competent larvae. (c) Average [log.sub.2] read count per gene per million reads mapped (CPM). (d) Differential expression test F-statistic. (e) Differential expression test P-value. (f) Benjamini-Hochberg-adjusted P-value to control type I error. FDR, false discovery rate. (g) InterPro annotation descriptions for each gene. Table A4 Polycystic kidney disease-like genes upregulaled in competent larvae Capitella teleta Gene ID (a) [Log.sub.2] FC (b) [Log.sub.2] CPM (c) F (d) CapteG169289 -1.41 7.96 72.56 CapteG119174 -2.06 5.11 111.96 CapteG89348 -2.17 2.54 58.06 CapteG185815 -2.39 5.43 108.51 CapteG89699 -2.90 5.12 53.23 Gene ID (a) P-value (e) FDR (F) InterPro descriptors (g) CapteG169289 2.34E-06 2.00E-04 Polycystic kidney disease type 2 protein CapteG119174 2.43E-07 4.77E-05 Polycystin cation channel, PKD1/PKD2 CapteG89348 7.18E-06 4.31E-04 Polycystin cation channel, PKD1/PKD2 CapteG185815 2.88E-07 5.27E-05 Polycystic kidney disease type 2 protein CapteG89699 1.10E-05 5.84E-04 Polycystic kidney disease type 2 protein (a) Ensembl Metazoa gene ID. (b) [Log.sub.2] fold change (FC) in gene expression between precompetent and competent larvae of C. teleta. Positive values are expressed more highly in precompetent larvae, while negative values are expressed more highly in competent larvae. (c) Average [log.sub.2] read count per gene per million reads mapped (CPM). (d) Differential expression test F-statistic. (e) Differential expression test P-value. (f) Benjamini-Hochberg-adjusted P-value to control type I error. FDR, false discovery rate. (g) InterPro annotation descriptions for each gene. Table A5 G-protein-coupled receptors upregulated in competent larvae of Capitella teleta Gene ID (a) [Log.sub.2] FC (b) [Log.sub.2] CPM (c) F (d) CapteG190026 -1.01 2.61 19.94 CapteG147802 -1.01 2.59 24.90 CapteG34777 -1.01 2.39 28.89 CapteG196014 -1.03 3.69 39.69 CapteG196564 -1.04 2.01 12.37 CapteG213150 -1.06 3.28 36.89 CapteG115578 -1.08 2.66 27.63 CapteG90908 -1.11 2.96 30.33 CapteG152907 -1.15 3.88 48.11 CapteG224577 -1.15 5.40 53.86 CapteG201805 -1.17 0.51 12.77 CapteG196425 -1.20 1.17 19.53 CapteG194428 -1.23 1.46 23.08 CapteG227407 -1.24 1.93 27.01 CapteG196555 -1.25 1.13 19.31 CapteG141683 -1.25 2.83 31.67 CapteG198894 -1.26 1.13 22.73 CapteG195782 -1.30 1.36 25.28 CapteG209584 -1.32 1.32 28.60 CapteG26928 -1.35 1.40 19.95 CapteG205658 -1.38 1.09 17.22 CapteG140881 -1.39 1.15 18.98 CapteG194422 -1.40 0.84 18.38 CapteG134768 -1.43 3.88 23.84 CapteG98913 -1.44 0.56 16.59 CapteG188220 -1.48 0.16 18.47 CapteG192959 -1.49 2.52 20.79 CapteG199664 -1.51 2.18 32.76 CaptcG215255 -1.53 2.03 20.90 CapteG110011 -1.60 1.28 46.07 CapteG213630 -1.61 1.81 36.12 CapteG216885 -1.75 3.24 35.03 CapteG145194 -1.76 2.26 64.37 CapteG192499 -1.76 1.72 50.85 CapteG131608 -1.97 -0.71 17.02 CapteG108341 -2.18 -1.54 11.92 CapteG199650 -2.40 -0.90 20.28 CapteG201394 -2.41 -1.40 13.26 CapteG19593.3 -2.45 -0.25 20.98 CapteG199130 -2.52 -1.18 22.55 CapteG193146 -2.90 -1.31 17.04 CapteG37194 -2.91 -1.47 17.86 CapteG20I967 -3.20 -1.54 16.79 CapteG205921 -3.35 5.01 28.48 Gene ID (a) P-value (e) FDR (f) InterPro descriptors (g) CapteG190026 1.38E-03 1.88E-02 GPCR, rhodopsin-like. 7TM CapteG147802 1.36E-03 1.86E-02 G-protein-coupled receptor, rhodopsin-like | galanin receptor family CapteG34777 1.64E-03 2.12E-02 G-protein-coupled receptor, rhodopsin-like | neuropeptide Y receptor family CapteG196014 2.21E-04 4.96E-03 GPCR, rhodopsin-like, 7TM CapteG196564 4.40E-03 4.31E-02 GPCR, rhodopsin-like. 7TM CapteG213150 2.54E-04 5.45E-03 GPCR, rhodopsin-like, 7TM CapteG115578 5.66E-04 9.77E-03 GPCR, rhodopsin-like, 7TM CapteG90908 2.47E-04 5.35E-03 G-protein-coupled receptor, rhodopsin-like | thyrotropin -releasing hormone receptor CapteG152907 2.77E-05 1.09E-03 GPCR, rhodopsin-like, 7TM CapteG224577 1.04E-05 5.66E-04 GPCR, rhodopsin-like, 7TM CapteG201805 3.98E-03 4.00E-02 GPCR, rhodopsin-like, 7TM CapteG196425 1.21E-03 1.72E-02 GPCR, family 3. [gamma]- aminobutyric acid receptor, type B CapteG194428 5.96E-04 1.02E-02 G-protein-coupled receptor, rhodopsin-like CapteG227407 2.96E-04 6.11E-03 GPCR, rhodopsin-like, 7TM CapteG196555 9.27E-04 1.41E-02 GPCR, rhodopsin-like, 7TM CapteG141683 1.22E-04 3.14E-03 G-protein-coupled receptor, rhodopsin-like | 5- hydroxytryptamine 6 receptor CapteG198894 7.51E-04 1.21E-02 GPCR, rhodopsin-like, 7TM CapteG195782 3.45E-04 6.84E-03 GPCR, rhodopsin-like, 7TM CapteG209584 3.12E-04 6.33E-03 GPCR, rhodopsin-like. 7TM CapteG26928 8.19E-04 1.29E-02 GPCR, rhodopsin-like. 7TM CapteG205658 1.42E-03 1.91E-02 GPCR. family 3, [gamma]- aminobutyric acid receptor, type B CapteG140881 9.91E-04 1.48E-02 GPCR, family 3, [gamma]- aminobutyric acid receptor, type B CapteG194422 1.12E-03 1.61E-02 GPCR. rhodopsin-like, 7TM CapteG134768 4.05E-04 7.65E-03 G-protein-coupled receptor, rhodopsin-like | glycoprotein hormone receptor family CapteG98913 1.63E-03 2.10E-02 GPCR, rhodopsin-like, 7TM CapteG188220 1.10E-03 1.59E-02 GPCR. rhodopsin-like. 7TM CapteG192959 6.98E-04 1.14E-02 GPCR. family 3. [gamma]- aminobutyric acid receptor, type B CapteG199664 1.05E-04 2.82E-03 GPCR. rhodopsin-like. 7TM CaptcG215255 6.84E-04 1.13E-02 GPCR. rhodopsin-like. 7TM CapteG110011 2.20E-05 9.30E-04 GPCR, rhodopsin-like, 7TM CapteG213630 6.81E-05 2.11E-03 GPCR. rhodopsin-like. 7TM CapteG216885 7.82E-05 2.31E-03 GPCR. rhodopsin-like. 7TM CapteG145194 4.30E-06 3.09E-04 GPCR. family 3. [gamma]- aminobutyricacid receptor, type B CapteG192499 1.37E-05 6.78E-04 GPCR, rhodopsin-like, 7TM CapteG131608 1.48E-03 1.97E-02 GPCR. rhodopsin-like, 7TM CapteG108341 4.96E-03 4.69E-02 GPCR, rhodopsin-like, 7TM CapteG199650 7.69E-04 1.23E-02 GPCR. rhodopsin-like, 7TM CapteG201394 3.52E-03 3.68E-02 GPCR, rhodopsin-like, 7TM CapteG19593.3 6.74E-04 l.llE-02 GPCR. rhodopsin-like. 7TM CapteG199130 5.07E-04 9.02E-03 GPCR, rhodopsin-like, 7TM CapteG193146 1.47E-03 1.96E-02 GPCR, rhodopsin-like, 7TM CapteG37194 1.24E-03 1.74E-02 GPCR, rhodopsin-like, 7TM CapteG20I967 1.56E-03 2.04E-02 GPCR, rhodopsin-like. 7TM CapteG205921 1.94E-04 4.48E-03 GPCR. rhodopsin-like, 7TM (a) Ensembl Metazoa gene ID. (b) [Log.sub.2] fold change (FC) in gene expression between precompetent and competent larvae of C. teleta. Positive values are expressed more highly in precompetent larvae, while negative values are expressed more highly in competent larvae. (c) Average [log.sub.2] read count per gene per million reads mapped (CPM). (d) Differential expression test F-statistic. (e) Differential expression test P-value. (f) Benjamini-Hochberg-adjusted P-value to control type I error. FDR. false discovery rate. (g) InterPro annotation descriptions for each gene. GPCR, G-protein-coupled receptor; TM, transmembrane (i.e., 7TM indicates that there are 7 transmembrane domains in the protein). Table A6 Other receptors upregulated in competent larvae of Capitella teleta Gene ID (a) [Log.sub.2] FC (b) [Log.sub.2] CPM (c) F (d) CapteG148064 -1.02 2.56 32.92 CapteG192796 -1.06 2.03 22.09 CapteG219746 -1.12 2.85 34.09 CapteG152072 -1.15 5.02 48.78 CapteG188571 -1.17 3.89 41.37 CapteG204388 -1.66 2.19 44.63 CapteG98250 -1.85 -0.67 17.42 CapteG204976 -2.25 -1.65 11.62 CapteG205451 -1.14 0.76 11.66 CapteG123873 -1.62 0.88 23.06 CapteG221712 -1.05 4.27 40.09 CapteG217687 -1.82 0.93 38.69 CapteG214390 -1.69 -0.57 12.10 Gene ID (a) P-value (e) FDR (f) InterPro descriptors (g) CapteG148064 1.25E-03 1.75E-02 [gamma]-Aminobutyric acid A receptor/glycine receptor [alpha] CapteG192796 1.69E-03 2.17E-02 Ionotropic glutamate receptor CapteG219746 2.64E-04 5.61E-03 Ionotropic glutamate receptor CapteG152072 1.68E-05 7.71E-04 Ionotropic glutamate receptor CapteG188571 3.65E-05 1.35E-03 Ionotropic glutamate receptor CapteG204388 2.56E-05 1.04E-03 Ionotropic glutamate receptor CapteG98250 1.36E-03 1.86E-02 Ionotropic glutamate receptor CapteG204976 5..36E-03 4.95E-02 Ionotropic glutamate receptor CapteG205451 5.3 IE-03 4.92E-02 Nicotinic acetylcholine-gated receptor, transmembrane domain CapteG123873 4.64E-04 8.47E-03 Nicotinic acetylcholine-gated receptor, transmembrane domain CapteG221712 5.92E-05 1.91E-03 Neurotransmitter-gated ion channel CapteG217687 4.97E-05 1.67E-03 Neurotransmitter-gated ion channel CapteG214390 4.73E-03 4.54E-02 Gustatory receptor (a) Ensembl Metazoa gene ID. (b) [Log.sub.2] fold change (FC) in gene expression between precompetent and competent larvae of C. teleta. Positive values are expressed more highly in precompetent larvae, while negative values are expressed more highly in competent larvae. (c) Average [log.sub.2] read count per gene per million reads mapped (CPM). (d) Differential expression test F-statistic. (e) Differential expression test P-value. (f) Benjamini-Hochberg-adjusted P-value to control type I error. FDR, false discovery rate. (g) InterPro annotation descriptions for each gene.
ROBERT BURNS (*) AND JAN PECHENIK
Department of Biology. Tufts University, Medford, Massachusetts 02155
Received 14 August 2016; Accepted 5 April 2017; Published online 14 August 2017.
(*) To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
Abbreviations: 5-[HT.sub.6], 5-hydroxytryptamine 6; ASW, artificial seawater; DEG/ENaC. degenerin/epithelial sodium channel; FDR. false discovery rate; GABA, [gamma]-aminobutyric acid; GPCR. G-protein-coupled receptor; HSP. heat-shock protein; NCBI. National Center for Biotechnology Information; NOS. nitric oxide synthase; PKD. polycystic kidney disease; UTR, untranslated region.
Table 1 Sequencing library quality control statistics after quality trimming and alignment Sequenced Reads remaining % cleaned % coding % UTR Sample reads after cleaning reads aligned bases bases Cl 28,767,983 28,458,569 95.1 57.94 14.83 C2 19,655,572 19,434,838 95.3 60.31 14.04 C3 21.763,337 21,464,080 95.2 52.64 15.40 C4 23,376,691 23,056,643 94.9 57.06 14.97 P1 25,878,108 25,278,205 95.5 59.04 13.82 P2 22,300,488 22,016,305 95.4 60.11 14.76 P3 29,055.316 28.743,172 95.8 66.58 11.98 P4 29,266.981 28,965,035 95.9 67.10 11.83 % mRNA % intronic % intergenic % rRNA Sample bases bases bases bases Cl 72.77 5.58 20.78 0.86 C2 74.35 5.23 19.88 0.55 C3 68.04 7.49 22.57 1.90 C4 72.03 5.50 21.81 0.66 P1 72.86 4.59 18.62 3.93 P2 74.87 5.00 19.28 0.85 P3 78.56 4.49 16.29 0,66 P4 78.92 4.46 15.74 0.87 The "% bases" columns represent the percentage of aligned bases that aligned to the given genomic region for that sample. Samples C1-C4 are replicate libraries of competent larvae, while samples P1-P4 are replicate libraries of precompetent larvae. UTR. untranslated region. Table 2 Gene ontologies (GO) enriched in precompetent larvae of Capitella teleta GO ID GO term GO:0006457 Protein folding GO:0004298 Threonine-type endopeptidase activity GO:0051082 Unfolded protein binding GO:0005524 ATP binding GO:0005852 Eukaryotic translation initiation factor 3 complex GO:0003743 Translation initiation factor activity GO:0019773 Proteasome core complex, [alpha]-subunit complex GO:0006270 DNA replication initiation GO:0000398 mRNA splicing, via spliceosome GO:0003755 Peptidyl-prolyl cis-trans isomerase activity GO:0000413 Protein peptidyl-prolyl isomerization GO:0006511 Ubiquitin-dependent protein catabolic process GO:0004579 Dolichyl-diphosphooligosaccharide-protein glycotransferase activity GO:0042555 MCM complex GO:0006779 Porphyrin-containing compound biosynthetic process GO:0045454 Cell redox homeostasis GO:0003678 DNA helicase activity GO:0015671 Oxygen transport GO:0005732 Small nucleolar ribonucleoprotein complex GO:0042176 Regulation of protein catabolic process GO:0022624 Proteasome accessory complex GO:0008250 Oligosaccharyltransferase complex GO:0005885 Arp2/3 protein complex GO:0016272 Prefoldin complex GO:0008641 Small-protein-activating enzyme activity GO:0006886 Intracellular protein transport GO:0016051 Carbohydrate biosynthetic process GO:0008017 Microtubule binding GO:0004814 Arginine-tRNA ligase activity GO:0005092 GDP-dissociation inhibitor activity GO:0006465 Signal peptide processing GO:0006420 Arginyl-tRNA aminoacylation GO:0006281 DNA repair GO:0008173 RNA methyltransferase activity GO:0020037 Heme binding GO:0006221 Pyrimidine nucleotide biosynthetic process GO:0000070 Mitotic sister chromatid segregation GO:0006739 NADP metabolic process GO:0031981 Nuclear lumen GO:0005871 Kinesin complex GO:0051920 Peroxiredoxin activity GO:0006890 Retrograde vesicle-mediated transport, Golgi to ER GO:0051087 Chaperone binding GO:0003918 DNA topoisoinerase type II (ATP-hydrolyzing) activity GO ID Type (a) No, of genes (b) FDR (c) GO:0006457 P 33 3.00E-19 GO:0004298 F 17 1.50E-I6 GO:0051082 F 20 1.70E-I2 GO:0005524 F 120 8.50E-I0 GO:0005852 C 10 1.60E-08 GO:0003743 F 13 1.30E-07 GO:0019773 C 7 2.40E-07 GO:0006270 P 7 1.90E-05 GO:0000398 P 11 2.10E-05 GO:0003755 F 7 3.40E-04 GO:0000413 P 7 3.40E-04 GO:0006511 P 15 4.70E-04 GO:0004579 F 4 5.90E-04 GO:0042555 C 5 6.80E-04 GO:0006779 P 6 1.40E-03 GO:0045454 P 12 1.50E-03 GO:0003678 F 10 1.70E-03 GO:0015671 P 11 5.40E-03 GO:0005732 C 5 5.80E-03 GO:0042176 P 3 7.50E-03 GO:0022624 C 3 7.50E-03 GO:0008250 C 3 7.50E-03 GO:0005885 C 4 1.30E-02 GO:0016272 C 4 1.30E-02 GO:0008641 F 5 1.40E-02 GO:0006886 P 24 1.50E-02 GO:0016051 P 14 1.90E-02 GO:0008017 F 12 2.30E-02 GO:0004814 F 3 2.30E-02 GO:0005092 F 3 2..30E-02 GO:0006465 P 3 2.30E-02 GO:0006420 P 3 2.30E-02 GO:0006281 P 22 2.40E-02 GO:0008173 F 6 2.60E-02 GO:0020037 F 26 2.70E-02 GO:0006221 P 6 3.30E-02 GO:0000070 P 6 3.30E-02 GO:0006739 P 4 3.50E-02 GO:0031981 C 15 4.80E-02 GO:0005871 C 10 4.90E-02 GO:0051920 F 3 4.90E-02 GO:0006890 P 3 4.90E-02 GO:0051087 F 3 4.90E-02 GO:0003918 F 3 4.90E-02 (a) GO category that each GO term is derived from (C, celltilar component; F, biological function; P. biological process). (b) Number of annotated, differentially expressed genes in the given GO. (c) Benjamini-Hochberg-corrected P-value as calculated by a Fisher's exact test. FDR. false discovery rate. Table 3 Gene ontologies (GO) enriched in competent larvae of Capitella teleta GO ID GO term GO:000.5272 Sodium channel activity GO:0006814 Sodium ion transport GO:0005509 Calcium ion binding GO:0016020 Membrane GO:0003840 [gamma]-Glutamyltransferase activity GO:0006749 Glutathione metabolic process GO:0004129 Cytochrome c oxidase activity GO:0080019 Fatty-acyl-CoA reductase (alcohol-forming) activity GO ID Type (a) No. of genes (b) FDR (c) GO:000.5272 F 24 3.20E-12 GO:0006814 P 25 1.30E-10 GO:0005509 F 45 1.30E-04 GO:0016020 C 174 1.40E-04 GO:0003840 F 5 2.40E-03 GO:0006749 P 6 1.40E-02 GO:0004129 F 5 1.60E-02 GO:0080019 F 4 1.60E-02 (a) GO category that each GO term is derived from (C, cellular component; F. biological function; P. biological process). (b) Number of annotated, differentially expressed genes in the given GO. (c) Benjamini-Hochberg-corrected P-value as calculated by a Fisher's exact test. FDR. false discovery rate.
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|Author:||Burns, Robert; Pechenik, Jan|
|Publication:||The Biological Bulletin|
|Date:||Jun 1, 2017|
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