Transcriptomic insights into the dominance of two phototrophs throughout the water column of a tropical hypersaline-alkaline crater lake (Dziani Dzaha, Mayotte).

Saline-alkaline lakes often shelter high biomasses despite challenging conditions, owing to the occurrence of highly adapted phototrophs. Dziani Dzaha (Mayotte) is one such lake characterized by the stable co-dominance of the cyanobacterium Limnospira platensis and the picoeukaryote Picocystis salinarum throughout its water column. Despite light penetrating only into the uppermost meter, the prevailing co-dominance of these species persists even in light- and oxygen-deprived zones. Here, a depth profile of phototrophs metatranscriptomes, annotated using genomic data from isolated strains, is employed to identify expression patterns of genes related to carbon processing pathways including photosynthesis, transporters and fermentation. The findings indicate a prominence of gene expression associated with photosynthesis, with a peak of expression around 1 m below the surface, although the light intensity is very low and only red and dark red wavelengths can reach it, given the very high turbidity linked to the high biomass of L. platensis. Experiments on strains confirmed that both species do grow under these wavelengths, at rates comparable to those obtained under white light. A decrease in the expression of photosynthesis-related genes was observed in L. platensis with increasing depth, whereas P. salinarum maintained a very high pool of psbA transcripts down to the deepest point as a possible adaptation against photodamage, in the absence and/or very low levels of expression of genes involved in protection. In the aphotic/anoxic zone, expression of genes involved in fermentation pathways suggests active metabolism of reserve or available dissolved carbon compounds. Overall, L. platensis seems to be adapted to the uppermost water layer, where it is probably maintained thanks to gas vesicles, as evidenced by high expression of the gvpA gene. In contrast, P. salinarum occurs at similar densities throughout the water column, with a peak in abundance and gene expression levels which suggests a better adaptation to lower light intensities. These slight differences may contribute to limited inter-specific competition, favoring stable co-dominance of these two phototrophs.

Genomes of nine biofilm-forming filamentous strains of Cyanobacteria (genera Jaaginema, Scytonema, and Karukerafilum gen. nov.) isolated from mangrove habitats of Guadeloupe (Lesser Antilles).

Biofilm-forming cyanobacteria are abundant in mangrove ecosystems, colonizing various niches including sediment surface and periphyton where they can cover large areas, yet have received limited attention. Several filamentous isolates were recently isolated from Guadeloupe, illustrating the diversity and novelty present in these biofilms. In this study, nine strains belonging to three novel lineages found abundantly in Guadeloupe biofilms are characterized by genome sequencing, morphological and ultrastructural examination, metabolome fingerprinting and searched for secondary metabolites biosynthesis pathways. Assignation of two lineages to known genera is confirmed, namely Scytonema and Jaaginema. The third lineage corresponds to a new Coleofasciculales genus herein described as Karukerafilum gen. nov. The four strains belonging to this genus group into two subclades, one of which displays genes necessary for nitrogen fixation as well as the complete pathway for geosmin production. This study gives new insights into the diversity of mangrove biofilm-forming cyanobacteria, including genome-based description of a new genus and the first genome sequence available for the genus Jaaginema.

Monospecific renaming within the cyanobacterial genus Limnospira (Spirulina) and consequences for food authorization.

AIMS: The cyanobacterial genus, Limnospira (anc. Arthrospira Stizenberger ex Gomont 1892), commonly called "Spirulina", is widely used for commercial purposes because of its high protein content and beneficial probiotic metabolites. Thus, the taxonomy of this genus is important because of its consequences for food applications. METHODS AND RESULTS: We constructed a database with formation on all Limnospira strains plus new ones from 72 new French isolates. We used a polyphasic approach (phylogenetic, phylogenomic, presence or absence of coding DNA sequences, morphological, and ultrastructure analyses) to confirm that the species A. platensis belonged to the genus Limnospira (L. platensis Gomont comb. nov. Basionym. Arthrospira platensis Gomont 1892) and that the genus Limnospira was monospecific, only represented by L. platensis. CONCLUSIONS: This study highlighted the large intra-specific diversity of L. platensis, independent of the affiliations of the phylogenetic clades or geographical location of the habitats and the subsequent physiological and metabolic plasticity.

Intra-population genomic diversity of the bloom-forming cyanobacterium, Aphanizomenon gracile, at low spatial scale.

Cyanobacteria are oxygenic photosynthetic bacteria that perform a substantial part of the global primary production. Some species are responsible for catastrophic environmental events, called blooms, which have become increasingly common in lakes and freshwater bodies as a consequence of global changes. Genotypic diversity is considered essential for marine cyanobacterial population, allowing it to cope with spatio-temporal environmental variations and to adapt to specific micro-niches in the ecosystem. This aspect is underestimated in the study of bloom development, however, and given little notice in studies of the ecology of harmful cyanobacteria. Here we compared the genomes of four strains of Aphanizomenon gracile, a species of filamentous toxinogenic cyanobacteria (Nostocales) found worldwide in fresh and brackish water. Millimeter-sized fascicles were isolated from a single water sample and have been maintained in culture since 2010. A comparative study revealed extensive heterogeneity in gene contents, despite similar genome size and high similarity indices. These variations were mainly associated with mobile genetic elements and biosynthetic gene clusters. For some of the latter, metabolomic analysis confirmed the production of related secondary metabolites, such as cyanotoxins and carotenoids, which are thought to play a fundamental role in the cyanobacterial fitness. Altogether, these results demonstrated that an A. gracile bloom could be a highly diverse population at low spatial scale and raised questions about potential exchanges of essential metabolites between individuals.

Disruption of fish gut microbiota composition and holobiont's metabolome during a simulated Microcystis aeruginosa (Cyanobacteria) bloom.

BACKGROUND: Cyanobacterial blooms are one of the most common stressors encountered by metazoans living in freshwater lentic systems such as lakes and ponds. Blooms reportedly impair fish health, notably through oxygen depletion and production of bioactive compounds including cyanotoxins. However, in the times of the "microbiome revolution", it is surprising that so little is still known regarding the influence of blooms on fish microbiota. In this study, an experimental approach is used to demonstrate that blooms affect fish microbiome composition and functions, as well as the metabolome of holobionts. To this end, the model teleost Oryzias latipes is exposed to simulated Microcystis aeruginosa blooms of various intensities in a microcosm setting, and the response of bacterial gut communities is evaluated in terms of composition and metabolome profiling. Metagenome-encoded functions are compared after 28 days between control individuals and those exposed to highest bloom level. RESULTS: The gut bacterial community of O. latipes exhibits marked responses to the presence of M. aeruginosa blooms in a dose-dependent manner. Notably, abundant gut-associated Firmicutes almost disappear, while potential opportunists increase. The holobiont's gut metabolome displays major changes, while functions encoded in the metagenome of bacterial partners are more marginally affected. Bacterial communities tend to return to original composition after the end of the bloom and remain sensitive in case of a second bloom, reflecting a highly reactive gut community. CONCLUSION: Gut-associated bacterial communities and holobiont functioning are affected by both short and long exposure to M. aeruginosa, and show evidence of post-bloom resilience. These findings point to the significance of bloom events to fish health and fitness, including survival and reproduction, through microbiome-related effects. In the context of increasingly frequent and intense blooms worldwide, potential outcomes relevant to conservation biology as well as aquaculture warrant further investigation. Video Abstract.

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis.

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.

Metagenome-Based Exploration of Bacterial Communities Associated with Cyanobacteria Strains Isolated from Thermal Muds.

Cyanobacteria constitute a pioneer colonizer of specific environments for whom settlement in new biotopes precedes the establishment of composite microbial consortia. Some heterotrophic bacteria constitute cyanobacterial partners that are considered as their cyanosphere, being potentially involved in mutualistic relationships through the exchange and recycling of key nutrients and the sharing of common goods. Several non-axenic cyanobacterial strains have been recently isolated, along with their associated cyanospheres, from the thermal mud of Balaruc-les-Bains (France) and the biofilms of the retention basin where they develop. The community structure and relationships among the members of the isolated cyanobacterial strains were characterized using a metagenomic approach combined with taxonomic and microscopic descriptions of the microbial consortia. The results provided insights into the potential role and metabolic capabilities of the microorganisms of thermal mud-associated cyanobacterial biofilms. Thus, the physical proximity, host-specificity, and genetic potential functions advocate for their complementarity between cyanobacteria and their associated microbiota. Besides these findings, our results also highlighted the great influence of the reference protein database chosen for performing functional annotation of the metagenomes from organisms of the cyanosphere and the difficulty of selecting one unique database that appropriately covers both autotroph and heterotroph metabolic specificities.

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.

Draft Genome Sequence of the Toxic Freshwater Microcystis aeruginosa Strain PMC 728.11 (Cyanobacteria, Chroococcales).

Microcystis aeruginosa is one of the major species that cause toxic cyanobacterial blooms in freshwater systems worldwide. Here, we report the draft genome sequence of M. aeruginosa PMC 728.11, a microcystin-producing cyanobacterium isolated from the freshwater reservoir of Juanon in Valence, France. The genome sequence contains 276 contigs, consisting of 5,536,025 bp and 5,594 putative protein-coding genes, among which are several biosynthetic gene clusters encoding enzyme complexes involved in the production of various bioactive and toxic metabolites.

Microbiome-Aware Ecotoxicology of Organisms: Relevance, Pitfalls, and Challenges.

Over the last 15 years, the advent of high-throughput "omics" techniques has revealed the multiple roles and interactions occurring among hosts, their microbial partners and their environment. This microbiome revolution has radically changed our views of biology, evolution, and individuality. Sitting at the interface between a host and its environment, the microbiome is a relevant yet understudied compartment for ecotoxicology research. Various recent works confirm that the microbiome reacts to and interacts with contaminants, with consequences for hosts and ecosystems. In this paper, we thus advocate for the development of a "microbiome-aware ecotoxicology" of organisms. We emphasize its relevance and discuss important conceptual and technical pitfalls associated with study design and interpretation. We identify topics such as functionality, quantification, temporality, resilience, interactions, and prediction as major challenges and promising venues for microbiome research applied to ecotoxicology.

Anti-Inflammatory, Antioxidant, and Wound-Healing Properties of Cyanobacteria from Thermal Mud of Balaruc-Les-Bains, France: A Multi-Approach Study.

Background: The Balaruc-les-Bains' thermal mud was found to be colonized predominantly by microorganisms, with cyanobacteria constituting the primary organism in the microbial biofilm observed on the mud surface. The success of cyanobacteria in colonizing this specific ecological niche can be explained in part by their taxa-specific adaptation capacities, and also the diversity of bioactive natural products that they synthesize. This array of components has physiological and ecological properties that may be exploited for various applications. Methods: Nine cyanobacterial strains were isolated from Balaruc thermal mud and maintained in the Paris Museum Collection (PMC). Full genome sequencing was performed coupled with targeted and untargeted metabolomic analyses (HPLC-DAD and LC-MS/MS). Bioassays were performed to determine antioxidant, anti-inflammatory, and wound-healing properties. Results: Biosynthetic pathways for phycobiliproteins, scytonemin, and carotenoid pigments and 124 metabolite biosynthetic gene clusters (BGCs) were characterized. Several compounds with known antioxidant or anti-inflammatory properties, such as carotenoids, phycobilins, mycosporine-like amino acids, and aeruginosins, and other bioactive metabolites like microginins, microviridins, and anabaenolysins were identified. Secretion of the proinflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8 appeared to be inhibited by crude extracts of Planktothricoides raciborskii PMC 877.14, Nostoc sp. PMC 881.14, and Pseudo-chroococcus couteii PMC 885.14. The extract of the Aliinostoc sp. PMC 882.14 strain was able to slightly enhance migration of HaCat cells that may be helpful in wound healing. Several antioxidant compounds were detected, but no significant effects on nitric oxide secretion were observed. There was no cytotoxicity on the three cell types tested, indicating that cyanobacterial extracts may have anti-inflammatory therapeutic potential without harming body cells. These data open up promising uses for these extracts and their respective molecules in drugs or thermal therapies.

Therapeutic Potential of a New Jumbo Phage That Infects Vibrio coralliilyticus, a Widespread Coral Pathogen.

Biological control using bacteriophages is a promising approach for mitigating the devastating effects of coral diseases. Several phages that infect Vibrio coralliilyticus, a widespread coral pathogen, have been isolated, suggesting that this bacterium is permissive to viral infection and is, therefore, a suitable candidate for treatment by phage therapy. In this study, we combined functional and genomic approaches to evaluate the therapeutic potential of BONAISHI, a novel V. coralliilyticus phage, which was isolated from the coral reef in Van Phong Bay (Vietnam). BONAISHI appears to be strictly lytic for several pathogenic strains of V. coralliilyticus and remains infectious over a broad range of environmental conditions. This candidate has an unusually large dsDNA genome (303 kb), with no genes that encode known toxins or implicated in lysogeny control. We identified several proteins involved in host lysis, which may offer an interesting alternative to the use of whole bacteriophages for controlling V. coralliilyticus. A preliminary therapy test showed that adding BONAISHI to an infected culture of Symbiodinium sp. cells reduced the impact of V. coralliilyticus on Symbiodinium sp. photosynthetic activity. This study showed that BONAISHI is able to mitigate V. coralliilyticus infections, making it a good candidate for phage therapy for coral disease.

Corallivory and the microbial debacle in two branching scleractinians.

The grazing activity by specific marine organisms represents a growing threat to the survival of many scleractinian species. For example, the recent proliferation of the corallivorous gastropod Drupella now constitutes a critical case in all South-East Asian waters. If the damaging effects caused by this marine snail on coral polyps are relatively well known, the indirect incidence of predation on coral microbial associates is still obscure and might also potentially impair coral health. In this study, we compared the main ecological traits of coral-associated bacterial and viral communities living in the mucus layer of Acropora formosa and Acropora millepora, of healthy and predated individuals (i.e., colonized by Drupella rugosa), in the Bay of Van Phong (Vietnam). Our results show a substantial impact of the gastropod on a variety of microbiological markers. Colonized corals harbored much more abundant and active epibiotic bacteria whose community composition shifted toward more pathogenic taxa (belonging to the Vibrionales, Clostridiales, Campylobacterales, and Alteromonadales orders), together with their specific phages. Viral epibionts were also greatly influenced by Drupella corallivory with spectacular modifications in their concentrations, life strategies, genotype richness, and diversity. Novel and abundant circular Rep-encoding ssDNA viruses (CRESS-DNA viruses) were detected and characterized in grazed corals and we propose that their occurrence may serve as indicator of the coral health status. Finally, our results reveal that corallivory can cause severe dysbiosis by altering virus-bacteria interactions in the mucus layer, and ultimately favoring the development of local opportunistic infections.

Highly divergent ancient gene families in metagenomic samples are compatible with additional divisions of life.

BACKGROUND: Microbial genetic diversity is often investigated via the comparison of relatively similar 16S molecules through multiple alignments between reference sequences and novel environmental samples using phylogenetic trees, direct BLAST matches, or phylotypes counts. However, are we missing novel lineages in the microbial dark universe by relying on standard phylogenetic and BLAST methods? If so, how can we probe that universe using alternative approaches? We performed a novel type of multi-marker analysis of genetic diversity exploiting the topology of inclusive sequence similarity networks. RESULTS: Our protocol identified 86 ancient gene families, well distributed and rarely transferred across the 3 domains of life, and retrieved their environmental homologs among 10 million predicted ORFs from human gut samples and other metagenomic projects. Numerous highly divergent environmental homologs were observed in gut samples, although the most divergent genes were over-represented in non-gut environments. In our networks, most divergent environmental genes grouped exclusively with uncultured relatives, in maximal cliques. Sequences within these groups were under strong purifying selection and presented a range of genetic variation comparable to that of a prokaryotic domain. CONCLUSIONS: Many genes families included environmental homologs that were highly divergent from cultured homologs: in 79 gene families (including 18 ribosomal proteins), Bacteria and Archaea were less divergent than some groups of environmental sequences were to any cultured or viral homologs. Moreover, some groups of environmental homologs branched very deeply in phylogenetic trees of life, when they were not too divergent to be aligned. These results underline how limited our understanding of the most diverse elements of the microbial world remains, and encourage a deeper exploration of natural communities and their genetic resources, hinting at the possibility that still unknown yet major divisions of life have yet to be discovered.

Studying genome heterogeneity within the arbuscular mycorrhizal fungal cytoplasm.

Although heterokaryons have been reported in nature, multicellular organisms are generally assumed genetically homogeneous. Here, we investigate the case of arbuscular mycorrhizal fungi (AMF) that form symbiosis with plant roots. The growth advantages they confer to their hosts are of great potential benefit to sustainable agricultural practices. However, measuring genetic diversity for these coenocytes is a major challenge: Within the same cytoplasm, AMF contain thousands of nuclei and show extremely high levels of genetic variation for some loci. The extent and physical location of polymorphism within and between AMF genomes is unclear. We used two complementary strategies to estimate genetic diversity in AMF, investigating polymorphism both on a genome scale and in putative single copy loci. First, we used data from whole-genome pyrosequencing of four AMF isolates to describe genetic diversity, based on a conservative network-based clustering approach. AMF isolates showed marked differences in genome-wide diversity patterns in comparison to a panel of control fungal genomes. This clustering approach further allowed us to provide conservative estimates of Rhizophagus spp. genomes sizes. Second, we designed new putative single copy genomic markers, which we investigated by massive parallel amplicon sequencing for two Rhizophagus irregularis and one Rhizophagus sp. isolates. Most loci showed high polymorphism, with up to 103 alleles per marker. This polymorphism could be distributed within or between nuclei. However, we argue that the Rhizophagus isolates under study might be heterokaryotic, at least for the putative single copy markers we studied. Considering that genetic information is the main resource for identification of AMF, we suggest that special attention is warranted for the study of these ecologically important organisms.

Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP) kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG) transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT) and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.

Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

EGN: a wizard for construction of gene and genome similarity networks.

BACKGROUND: Increasingly, similarity networks are being used for evolutionary analyses of molecular datasets. These networks are very useful, in particular for the analysis of gene sharing, lateral gene transfer and for the detection of distant homologs. Currently, such analyses require some computer programming skills due to the limited availability of user-friendly freely distributed software. Consequently, although appealing, the construction and analyses of these networks remain less familiar to biologists than do phylogenetic approaches. RESULTS: In order to ease the use of similarity networks in the community of evolutionary biologists, we introduce a software program, EGN, that runs under Linux or MacOSX. EGN automates the reconstruction of gene and genome networks from nucleic and proteic sequences. EGN also implements statistics describing genetic diversity in these samples, for various user-defined thresholds of similarities. In the interest of studying the complexity of evolutionary processes affecting microbial evolution, we applied EGN to a dataset of 571,044 proteic sequences from the three domains of life and from mobile elements. We observed that, in Borrelia, plasmids play a different role than in most other eubacteria. Rather than being genetic couriers involved in lateral gene transfer, Borrelia's plasmids and their genes act as private genetic goods, that contribute to the creation of genetic diversity within their parasitic hosts. CONCLUSION: EGN can be used for constructing, analyzing, and mining molecular datasets in evolutionary studies. The program can help increase our knowledge of the processes through which genes from distinct sources and/or from multiple genomes co-evolve in lineages of cellular organisms.

Sequence comparative analysis using networks: software for evaluating de novo transcript assembly from next-generation sequencing.

DNA sequencing technology is becoming more accessible to a variety of researchers as costs continue to decline. As researchers begin to sequence novel transcriptomes, most of these data sets lack a reference genome and will have to rely on de novo assemblers. Making comparisons across assemblies can be difficult: each program has its strengths and weaknesses, and no tool exists to comparatively evaluate these data sets. We developed software in R, called Sequence Comparative Analysis using Networks (SCAN), to perform statistical comparisons between distinct assemblies. SCAN uses a reference data set to identify the most accurate de novo assembly and the "good" transcripts in the user's data. We tested SCAN on three publicly available transcriptomes, each assembled using three assembly programs. Moreover, we sequenced the transcriptome of the oomycete Achlya hypogyna and compared de novo assemblies from Velvet, ABySS, and the CLC Genomics Workbench assembly algorithms. One thousand one hundred twenty-eight of the CLC transcripts were statistically similar to the reference, compared with 49 of the Velvet transcripts and 937 of the ABySS transcripts. SCAN's strength is providing statistical support for transcript assemblies in a biological context. However, SCAN is designed to compare distinct node sets in networks, therefore it can also easily be extended to perform statistical comparisons on any network graph regardless of what the nodes represent.