Transcriptome Shotgun Assembly Sequences Research Articles

Dataset of de novo assembly and functional annotation of the transcriptomes of three native oleaginous microalgae from the Peruvian Amazon.

Microalgae are photosynthetic organisms with cosmopolitan distribution (i.e., marine, freshwater and terrestrial habitats) and possess a great diversity of species [1] and consequently an immense variation in biochemical compositions [2]. To date genomic information is available mainly from the model green microalga Chlamydomonas reinhardtii[3]. Here we provide the dataset of a de novo assembly and functional annotation of the transcriptomes of three native oleaginous microalgae from the Peruvian Amazon. Native oleaginous microalgae species Ankistrodesmus sp., Chlorella sp., and Scenedesmus sp. were cultured in triplicate using Chu-10 medium with or without a source of nitrate (NaNO3). Total RNA was purified, the cDNA libraries were constructed and sequenced as paired-end reads on an Illumina HiSeq™2500 platform. Transcriptomes were de novo assembled using Trinity v2.9.1. A total of 48,554 transcripts (range from 250 to 7966 bp; N50 = 1047) for Ankistrodesmus sp., 108,126 transcripts (range from 250 to 8160 bp; N50 = 1090) for Chlorella sp., and 77,689 transcripts (range from 250 to 8481 bp; N50 = 1281) for Scenedesmus sp. were de novo assembled. Completeness of the assembled transcriptomes were evaluated with the Benchmarking Universal Single-Copy Orthologs (BUSCO) software v2/v3. Functional annotation of the assembled transcriptomes was conducted with TransDecoder v3.0.1 and the web-based platforms Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS) and FunctionAnnotator. The raw reads were deposited into NCBI and are accessible via BioProject accession number PRJNA628966 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA628966) and Sequence Read Archive (SRA) with accession numbers: SRX8295665 (https://www.ncbi.nlm.nih.gov/sra/SRX8295665), SRX8295666 (https://www.ncbi.nlm.nih.gov/sra/SRX8295666), SRX8295667 (https://www.ncbi.nlm.nih.gov/sra/SRX8295667), SRX8295668 (https://www.ncbi.nlm.nih.gov/sra/SRX8295668), SRX8295669 (https://www.ncbi.nlm.nih.gov/sra/SRX8295669), and SRX8295670 (https://www.ncbi.nlm.nih.gov/sra/SRX8295670). Additionally, transcriptome shotgun assembly sequences and functional annotations are available via Discover Mendeley Data (https://data.mendeley.com/datasets/47wdjmw9xr/1).

Dataset of de novo assembly and functional annotation of the transcriptome during germination and initial growth of seedlings of Myrciaria Dubia "camu-camu".

Myrciaria dubia “camu-camu” is a native shrub of the Amazon that is commonly found in areas that are flooded for three to four months during the annual hydrological cycle. This plant species is exceptional for its capacity to biosynthesize and accumulate important quantities of a variety of health-promoting phytochemicals, especially vitamin C [1], yet few genomic resources are available [2]. Here we provide the dataset of a de novo assembly and functional annotation of the transcriptome from a pool of samples obtained from seeds during the germination process and seedlings during the initial growth (until one month after germination). Total RNA/mRNA was purified from different types of plant materials (i.e., imbibited seeds, germinated seeds, and seedlings of one, two, three, and four weeks old), pooled in equimolar ratio to generate the cDNA library and RNA paired-end sequencing was conducted on an Illumina HiSeq™2500 platform. The transcriptome was de novo assembled using Trinity v2.9.1 and SuperTranscripts v2.9.1. A total of 21,161 transcripts were assembled ranging in size from 500 to 10,001 bp with a N50 value of 1,485 bp. Completeness of the assembly dataset was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) software v2/v3. Finally, the assembled transcripts were functionally annotated using TransDecoder v3.0.1 and the web-based platforms Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS), and FunctionAnnotator. The raw reads were deposited into NCBI and are accessible via BioProject accession number PRJNA615000 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615000) and Sequence Read Archive (SRA) with accession number SRX7990430 (https://www.ncbi.nlm.nih.gov/sra/SRX7990430). Additionally, transcriptome shotgun assembly sequences and functional annotations are available via Discover Mendeley Data (https://data.mendeley.com/datasets/2csj3h29fr/1).

Prediction and analysis of GH14 family β-amylases in oat seedling extract: Structure and function insights using in silico approaches

Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify β-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as β-amylases. Insights into structural characterization of the oat predicted β-amylases were analyzed using in silico approaches. The identified β-amylases conserved the two Glu residues assigned as the “putative” catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (β/α)8-barrel architecture was found in the predicted oat β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)8-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat β-amylases and thus confirming that are exo-acting hydrolases.The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.

Proteomics and antivenomics of Echis carinatus carinatus venom: Correlation with pharmacological properties and pathophysiology of envenomation

The proteome composition of Echis carinatus carinatus venom (ECV) from India was studied for the first time by tandem mass spectrometry analysis. A total of 90, 47, and 22 distinct enzymatic and non-enzymatic proteins belonging to 15, 10, and 6 snake venom protein families were identified in ECV by searching the ESI-LC-MS/MS data against non-redundant protein databases of Viperidae (taxid 8689), Echis (taxid 8699) and Echis carinatus (taxid 40353), respectively. However, analysis of MS/MS data against the Transcriptome Shotgun Assembly sequences (87 entries) of conger E. coloratus identified only 14 proteins in ECV. Snake venom metalloproteases and snaclecs, the most abundant enzymatic and non-enzymatic proteins, respectively in ECV account for defibrinogenation and the strong in vitro pro-coagulant activity. Further, glutaminyl cyclase, aspartic protease, aminopeptidase, phospholipase B, vascular endothelial growth factor, and nerve growth factor were reported for the first time in ECV. The proteome composition of ECV was well correlated with its biochemical and pharmacological properties and clinical manifestations observed in Echis envenomed patients. Neutralization of enzymes and pharmacological properties of ECV, and immuno-cross-reactivity studies unequivocally point to the poor recognition of <20 kDa ECV proteins, such as PLA2, subunits of snaclec, and disintegrin by commercial polyvalent antivenom.

Polycipiviridae: a proposed new family of polycistronic picorna-like RNA viruses.

Solenopsis invicta virus 2 is a single-stranded positive-sense picorna-like RNA virus with an unusual genome structure. The monopartite genome of approximately 11 kb contains four open reading frames in its 5′ third, three of which encode proteins with homology to picornavirus-like jelly-roll fold capsid proteins. These are followed by an intergenic region, and then a single long open reading frame that covers the 3′ two-thirds of the genome. The polypeptide translation of this 3′ open reading frame contains motifs characteristic of picornavirus-like helicase, protease and RNA-dependent RNA polymerase domains. An inspection of public transcriptome shotgun assembly sequences revealed five related apparently nearly complete virus genomes isolated from ant species and one from a dipteran insect. By high-throughput sequencing and in silico assembly of RNA isolated from Solenopsis invicta and four other ant species, followed by targeted Sanger sequencing, we obtained nearly complete genomes for four further viruses in the group. Four further sequences were obtained from a recent large-scale invertebrate virus study. The 15 sequences are highly divergent (pairwise amino acid identities of as low as 17 % in the non-structural polyprotein), but possess the same overall polycistronic genome structure, which is distinct from all other characterized picorna-like viruses. Consequently, we propose the formation of a new virus family, Polycipiviridae, to classify this clade of arthropod-infecting polycistronic picorna-like viruses. We further propose that this family be divided into three genera: Chipolycivirus (2 species), Hupolycivirus (2 species) and Sopolycivirus (11 species), with members of the latter infecting ants in at least 3 different subfamilies.

Workflow and web application for annotating NCBI BioProject transcriptome data.

The volume of transcriptome data is growing exponentially due to rapid improvement of experimental technologies. In response, large central resources such as those of the National Center for Biotechnology Information (NCBI) are continually adapting their computational infrastructure to accommodate this large influx of data. New and specialized databases, such as Transcriptome Shotgun Assembly Sequence Database (TSA) and Sequence Read Archive (SRA), have been created to aid the development and expansion of centralized repositories. Although the central resource databases are under continual development, they do not include automatic pipelines to increase annotation of newly deposited data. Therefore, third-party applications are required to achieve that aim. Here, we present an automatic workflow and web application for the annotation of transcriptome data. The workflow creates secondary data such as sequencing reads and BLAST alignments, which are available through the web application. They are based on freely available bioinformatics tools and scripts developed in-house. The interactive web application provides a search engine and several browser utilities. Graphical views of transcript alignments are available through SeqViewer, an embedded tool developed by NCBI for viewing biological sequence data. The web application is tightly integrated with other NCBI web applications and tools to extend the functionality of data processing and interconnectivity. We present a case study for the species Physalis peruviana with data generated from BioProject ID 67621. Database URL: http://www.ncbi.nlm.nih.gov/projects/physalis/

Development of transcriptome shotgun assembly-derived markers in bunching onion (Allium fistulosum)

Bunching onion (Allium fistulosum L.) is one of the most important vegetables in Japan. Although expressed sequence tag (EST)-derived markers for bulb onion (A. cepa L.) have been developed from medium-scale sequencing, comparable EST sequences in bunching onion are lacking. In this study, we obtained 54,903 bunching onion unigenes using transcriptome shotgun assembly (TSA) and two next-generation sequencing technologies, GS-FLX and HiSeq 2000. When bunching onion and bulb onion unigenes were compared, 10,688 were estimated as reciprocal best-hit relationships. In the bunching onion TSA sequences, we discovered 2,396 di- to pentanucleotide simple sequence repeat (SSR) motifs and 5,505 exon–intron boundary sites. Moreover, we detected 9,002 single nucleotide polymorphisms and 4,335 insertion–deletion (InDel) by comparing sequence reads obtained from two inbred lines, “F” and “A.” TSA-derived SSR, cleaved amplified polymorphic sequences, InDels and intron-spanning markers were used to develop a linkage map. The genetic map, designated the FA map, contained 17 linkage groups with 364 markers (190 bunching onion TSAs, 96 bunching onion genomic SSRs, 39 bulb onion ESTs and 4 other markers) and covered a distance of 1,150 cM.

Large-scale development of EST-SSR markers in sponge gourd via transcriptome sequencing

Sponge gourd is a versatile vegetable that can be used as a household cleaning product, industrial material and medicine, and possesses anti-human immunodeficiency virus activity. In the current study, a large dataset composed of 77,858 unigenes derived from the sponge gourd transcriptome was assembled. Of all unigenes, 52,113 (66.93 %) had significant similarity to known proteins from NCBI non-redundant, Swiss-Prot, Clusters of Orthologous Group and Kyoto Encyclopedia of Genes and Genomes databases. Based on these generated sequences, we identified 12,932 putative simple sequence repeats (SSRs) and successfully designed 8,523 high-quality SSR primer pairs. Six hundred and forty-one primer pairs were randomly selected to be verified among S1174 [Luffa acutangula (L.) Roxb.], 93075 [L. cylindrica (L.) Roem.] and their hybrid F1. The result showed that 494 (77.07 %) exhibited successful amplification, of which 201 (40.69 %) revealed polymorphism between S1174 and 93075. Fifty polymorphic expressed sequence tag (EST)-SSRs were used to genotype 60 sponge gourd accessions. Cluster analysis revealed two major clusters, one comprising 42 accessions of L. acutangula and the other 18 accessions belonging to L. cylindrica. Transferability of the 77 polymorphic EST-SSR markers was investigated in six other cucurbits: pumpkin, cucumber, wax gourd, bitter gourd, bottle gourd and chieh-qua, and the percentage of cross-genus amplification was 85.90, 83.33, 83.33, 79.49, 75.64 and 74.36 %, respectively. This indicated that EST-SSRs in sponge gourd could well be applied to other cucurbits. These transcriptome shotgun assembly sequences and EST-SSR data are of great value for the discovery of novel genes and for marker-assisted selection in sponge gourd.

Identification of the first neuropeptides from the Amphipoda (Arthropoda, Crustacea)

Despite being used as models in the field of ecotoxicology, including use in studies of endocrine disruption, little is known about the hormonal systems of amphipods, particularly their peptidergic signaling systems. Here, transcriptome shotgun assembly (TSA) sequences were used to predict the structures of the first neuropeptides from members of this crustacean order. Using a well-established workflow, BLAST searches of the extant amphipod TSA data were conducted for putative peptide-encoding transcripts. The pre/preprohormones deduced from the identified TSA sequences were then used to predict the mature structures of amphipod neuropeptides. In total, 43 putative peptide-encoding transcripts were identified from three amphipods, Echinogammarus veneris, Hyalella azteca and Melita plumulosa. Collectively, 139 distinct mature peptides (110 from E. veneris alone) were predicted from these TSA sequences. The identified peptides included members of the adipokinetic hormone/red pigment concentrating hormone, allatostatin A, allatostatin B, allatostatin C, bursicon α, bursicon β, crustacean hyperglycemic hormone, diuretic hormone 31, FLRFamide, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone (PDH), proctolin, RYamide, SIFamide, sulfakinin and tachykinin-related peptide families. Of particular note were the identifications of orcokinins possessing SFDEIDR– rather than the typical NFDEIDR– amino-termini, e.g. SFDEINRSNFGFN, a carboxyl-terminally amidated orcokinin, i.e. SFDEINRSNFGFSamide, PDHs longer than the stereotypical 18 amino acids, e.g. NSELLNTLLGSKSLAALRAAamide, and a 13 rather than 12 amino acid long SIFamide, i.e. GPYRKPPFNGSIFamide. These data not only provide the first descriptions of native amphipod neuropeptides, but also represent a new resource for initiating investigations of peptidergic signaling in the Amphipoda.

In silico characterization of the peptidome of the sea louse Caligus rogercresseyi (Crustacea, Copepoda)

Copepods of the order Siphonostomatoida are a major concern for commercial aquaculture as many farmed fish serve as hosts for these parasitic crustaceans. Caligus rogercresseyi, a member of the Siphonostomatoida, is a significant problem for salmonid aquaculture in the Southern Hemisphere, and as such, a search for methods for controlling infestations of it is ongoing. One possibility for biological control of this and other copepod ectoparasites is endocrine manipulation. However, little is known about the native endocrine signaling systems in these animals. As part of an ongoing effort to characterize crustacean ectoparasite peptidergic systems, the publicly accessible C. rogercresseyi transcriptome shotgun assembly (TSA) was mined for peptide-encoding transcripts. Using the identified TSA sequences, precursor proteins were deduced and their mature peptides predicted. Thirty-three peptide-encoding transcripts were identified within the Caligus TSA dataset, with the structures of 131 distinct peptides characterized from the deduced pre/preprohormones. The predicted peptides included isoforms of allatostatin A, allatostatin B, bursicon α, bursicon β, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone, diuretic hormone 31, DXXRLamide, FLRFamide, FXGGXamide, GSEFLamide, insulin-like peptide (ILP), intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F (NPF), orcokinin and tachykinin-related peptide. The predicted ILPs are of particular note as they are the first members of this peptide family identified from a copepod. Similarly, the predicted complement of four distinct NPFs is larger than that known from other crustaceans. Taken collectively, these data greatly expand the known C. rogercresseyi peptidome and provide a foundation for initiating studies of peptidergic control in this species.

Prediction of the first neuropeptides from a member of the Remipedia (Arthropoda, Crustacea)

The Remipedia is a small, recently described crustacean class that inhabits submerged marine/anchialine cave systems. Phylogenetic and morphological investigations support a sister group relationship between these animals and the hexapods. The recent deposition of numerous (>100,000) transcriptome shotgun assembly sequences for Speleonectes cf. tulumensis provides a unique resource to identify proteins of interest from a member of the Remipedia. Here, this dataset was mined for sequences encoding putative neuropeptide pre/preprohormones, with the mature peptides predicted from the deduced precursors using an established workflow. The structures of 40 mature peptides were obtained via this strategy, including members of 11 well-known arthropod peptide families (adipokinetic hormone/corazonin-like peptide [ACP], allatostatin A, allatostatin C, diuretic hormone 31, eclosion hormone, ion transport peptide/crustacean hyperglycemic hormone, neuropeptide F, proctolin, SIFamide, sulfakinin and tachykinin-related peptide); these are the only peptides thus far described from any member of the Remipedia. Comparison of the Speleonectes isoforms with those from other crustaceans and hexapods revealed the peptidome of this species to have characteristics of both subphyla (e.g. it possesses the stereotypical decapod crustacean SIFamide and tachykinin-related peptide isoforms, while simultaneously being the only crustacean with an insect AKC). Moreover, BLAST searches in which the deduced Speleonectes precursors were compared to the pancrustacean protein database most frequently returned insect homologs as the closest matches. The peptidomic analyses presented here are consistent with the hypothesized phylogenetic position of the Remipedia within the Pancrustacea, and serve as a foundation from which to launch future investigations of peptidergic signaling in remipedes.

Coming out of the Shell: Building the Molecular Infrastructure for Research on Parasite-Harbouring Snails

Coming out of the Shell: Building the Molecular Infrastructure for Research on Parasite-Harbouring Snails

De Novo Sequencing and Transcriptome Analysis of the Central Nervous System of Mollusc Lymnaea stagnalis by Deep RNA Sequencing

The pond snail Lymnaea stagnalis is among several mollusc species that have been well investigated due to the simplicity of their nervous systems and large identifiable neurons. Nonetheless, despite the continued attention given to the physiological characteristics of its nervous system, the genetic information of the Lymnaea central nervous system (CNS) has not yet been fully explored. The absence of genetic information is a large disadvantage for transcriptome sequencing because it makes transcriptome assembly difficult. We here performed transcriptome sequencing for Lymnaea CNS using an Illumina Genome Analyzer IIx platform and obtained 81.9 M of 100 base pair (bp) single end reads. For de novo assembly, five programs were used: ABySS, Velvet, OASES, Trinity and Rnnotator. Based on a comparison of the assemblies, we chose the Rnnotator dataset for the following blast searches and gene ontology analyses. The present dataset, 116,355 contigs of Lymnaea transcriptome shotgun assembly (TSA), contained longer sequences and was much larger compared to the previously reported Lymnaea expression sequence tag (EST) established by classical Sanger sequencing. The TSA sequences were subjected to blast analyses against several protein databases and Aplysia EST data. The results demonstrated that about 20,000 sequences had significant similarity to the reported sequences using a cutoff value of 1e-6, and showed the lack of molluscan sequences in the public databases. The richness of the present TSA data allowed us to identify a large number of new transcripts in Lymnaea and molluscan species.

Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles.

Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately after fertilization in utero, before pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris' life cycle and parasitism. The transcriptome assembly has been submitted to NCBI Transcriptome Shotgun Assembly Sequence Database(http://www.ncbi.nlm.nih.gov/genbank/TSA.html) under accession numbers JI163767–JI182837 and JI210738–JI257410.