Transcript Contigs Research Articles

Composting reduces the risks of resistome in beef cattle manure at the transcriptional level.

Transcriptomic evidence is needed to determine whether composting is more effective than conventional stockpiling in mitigating the risk of resistome in livestock manure. The objective of this study is to compare composting and stockpiling for their effectiveness in reducing the risk of antibiotic resistance in beef cattle manure. Samples collected from the center and the surface of full-size manure stockpiling and composting piles were subject to metagenomic and metatranscriptomic analyses. While the distinctions in resistome between stockpiled and composted manure were not evident at the DNA level, the advantages of composting over stockpiling were evident at the transcriptomic level in terms of the abundance of antibiotic resistance genes (ARGs), the number of ARG subtypes, and the prevalence of high-risk ARGs (i.e., mobile ARGs associated with zoonotic pathogens). DNA and transcript contigs show that the pathogen hosts of high-risk ARGs included Escherichia coli O157:H7 and O25b:H4, Klebsiella pneumoniae, and Salmonella enterica. Although the average daily temperatures for the entire composting pile exceeded 55°C throughout the field study, more ARG and ARG transcripts were removed at the center of the composting pile than at the surface. This work demonstrates the advantage of composting over stockpiling in reducing ARG risk in active populations in beef cattle manure.IMPORTANCEProper treatment of manure before land application is essential to mitigate the spread of antibiotic resistance in the environment. Stockpiling and composting are two commonly used methods for manure treatment. However, the effectiveness of composting in reducing antibiotic resistance in manure has been debated. This work compared the ability of these two methods to reduce the risk of antibiotic resistance in beef cattle manure. Our results demonstrate that composting reduced more high-risk resistance genes at the transcriptomic level in cattle manure than conventional stockpiling. This finding not only underscores the effectiveness of composting in reducing antibiotic resistance in manure but also highlights the importance of employing RNA analyses alongside DNA analyses.

Isolation and Expression of Transcription Factors Involved in Somatic Embryo Development by Transcriptome Analysis of Embryogenic Callus of Thuja koraiensis

Thuja koraiensis Nakai (Cupressaceae) is an endangered and ecologically important conifer endemic to Korea. Previously, we established a protocol for micropropagation in T. koraiensis, which involved somatic embryogenesis from embryogenic callus of T. koraiensis. However, the molecular mechanisms underlying somatic embryogenesis remain unclear. Herein, we performed transcriptomic analysis to identify somatic embryogenesis-related genes of T. koraiensis via Illumina RNA sequencing. We conducted de novo transcriptome assembly using a Trinity assembler, which produced 274,077 transcript contigs clustered into 205,843 transcripts (unigenes), with an average length of 825 base pairs. Of all the unigenes, 14.69%, 18.62%, and 7.4% had homologs in the Gene Ontology, NCBI Non-redundant Protein, and NCBI Nucleotide databases, respectively. Among these mRNA sequences, expression of putative embryogenesis-associated transcription factors, namely BABYBOOM (BBM), WUSCHEL-RELATED HOMEOBOX (WOX), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK), was analyzed during somatic embryogenesis. RT-PCR analysis revealed that TkBBM, TkWOX, and TkSERK were highly expressed in embryogenic callus and seedling roots, whereas their expression was reduced in seedling leaves. Our findings provide new insights into the roles of BBM, WOX, and SERK in somatic embryogenesis. Our results may serve as a reference for comparative transcriptome analysis in related species and further aid functional genomics studies in conifers.

Meta-Transcriptomic Analysis of RNAseq Data Reveals Pacu and Loach Fish with Unusually High Levels of Myoglobin Expression in Skeletal Muscles.

Simple SummaryOxygen-binding proteins that mediate oxygen-binding for storage and consumption, to reduce energy, are very diverse in fish, depending on their habitats. In the present study, oxygen-binding protein gene expression in the skeletal muscle of 25 diverse fish species was examined by a meta-transcriptomic approach. By using RNAseq data, this is the first study to examine the high level of myoglobin, one of the oxygen-binding proteins, transcripts in pacu and loach fish that might be related to their high tolerance for the oxygen-deficient environment. In addition, this study presents the power of the current method to compare the fish oxygen-binding protein expression and its putative gene expansion event.Oxygen-binding proteins, such as myoglobin, hemoglobin, neuroglobin, and cytoglobin, play a role in oxygen binding and delivery to tissues. In icefish, the loss of myoglobin and hemoglobin genes has been reported to be an adaptive evolution event. This interesting finding prompted us to exam oxygen-binding protein expression in diverse fish species. Taking advantage of substantial RNAseq data deposited in the NCBI (National Center for Biotechnology Information) database, we adopted a meta-transcriptomic approach to explore and compare four oxygen-binding protein gene expression levels in the skeletal muscle of 25 diverse fish species for the first time. RNAseq data were downloaded from the NCBI Sequence Read Archive (SRA) database, and de novo assembly was performed to generate transcript contigs. The genes encoding oxygen-binding proteins were then identified by the BLAST search, and the relative expression level of oxygen-binding protein genes was normalized by the RPKM (Reads per Kilobase Million) method. By performing expression profiling, hierarchy clustering, and principal component analysis, pacu and loach fish were noticed by their high myoglobin expression levels in skeletal muscle tissues among 25 diverse fish species. In conclusion, we demonstrated that meta-transcriptomic analysis of RNAseq data is an informative approach to compare the oxygen-binding protein expression and putative gene expansion event in fish.

De novo transcriptome analysis of halotolerant bacterium Staphylococcus sp. strain P-TSB-70 isolated from East coast of India: In search of salt stress tolerant genes.

In the present study, we identified salt stress tolerant genes from the marine bacterium Staphylococcus sp. strain P-TSB-70 through transcriptome sequencing. In favour of whole-genome transcriptome profiling of Staphylococcus sp. strain P-TSB-70 (GenBank Accn. No. KP117091) which tolerated upto 20% NaCl stress, the strain was cultured in the laboratory condition with 20% NaCl stress. Transcriptome analyses were performed by SOLiD4.0 sequencing technology from which 10280 and 9612 transcripts for control and treated, respectively, were obtained. The coverage per base (CPB) statistics were analyzed for both the samples. Gene ontology (GO) analysis has been categorized at varied graph levels based on three primary ontology studies viz. cellular components, biological processes, and molecular functions. The KEGG analysis of the assembled transcripts using KAAS showed presumed components of metabolic pathways which perhaps implicated in diverse metabolic pathways responsible for salt tolerance viz. glycolysis/gluconeogenesis, oxidative phosphorylation, glutathione metabolism, etc. further involving in salt tolerance. Overall, 90 salt stress tolerant genes were identified as of 186 salt-related transcripts. Several genes have been found executing normally in the TCA cycle pathway, integral membrane proteins, generation of the osmoprotectants, enzymatic pathway associated with salt tolerance. Recognized genes fit diverse groups of salt stress genes viz. abc transporter, betaine, sodium antiporter, sodium symporter, trehalose, ectoine, and choline, that belong to different families of genes involved in the pathway of salt stress. The control sample of the bacterium showed elevated high proportion of transcript contigs (29%) while upto 20% salt stress treated sample of the bacterium showed a higher percentage of transcript contigs (31.28%). A total of 1,288 and 1,133 transcript contigs were measured entirely as novel transcript contigs in both control and treated samples, respectively. The structure and function of 10 significant salt stress tolerant genes of Staphylococcus sp. have been analyzed in this study. The information acquired in the present study possibly used to recognize and clone the salt stress tolerant genes and support in developing the salt stress-tolerant plant varieties to expand the agricultural productivity in the saline system.

De novo transcriptome assembly of a facultative parasitic nematode Pelodera (syn. Rhabditis) strongyloides

Pelodera strongyloides is a generally free-living gonochoristic facultative nematode. The whole genomic sequence of P. strongyloides remains unknown but 4 small subunit ribosomal RNA (ssrRNA) gene sequences are available. This project launched a de novo transcriptome assembly with 100 bp paired-end RNA-seq reads from normal, starved and wet-plate cultured animals. Trinity assembly tool generated 104,634 transcript contigs with N50 contig being 2195 bp and average contig length at 1103 bp. Transcriptome BLASTX matching results of five nematodes (C. elegans, Strongyloides stercoralis, Necator americanus, Trichuris trichiura, and Pristionchus pacificus) were consistent with their evolutionary relationships. Sixteen genes were identified to be homologous to key elements of the C. elegans RNA interference system, such as Dicer, Argonaute, RNA-dependent RNA polymerase and double strand RNA transport proteins. In starved samples, we observed up-regulation of cuticle related genes and 3 dauer formation genes. Dauer morphology was captured with enlarged phasmid under light microscopy, and dauer and normal larvae counts in clumps had a Pearson's product-moment correlation of 0.805 with P-value = 0.0088. Our results demonstrate that P. strongyloides could be used for studying nematode-related human or pet parasitic diseases. The sequenced assembled transcriptome reported here may be useful to understand the evolution of parasitism in Nematoda.

De novo whole transcriptome profiling of Edwardsiella tarda isolated from infected fish (Labeo catla)

Edwardsiella tarda belongs to the genera of Gram negative bacterium mainly associated with edwardsiellosis, the most commonly found infectious fish disease throughout the globe. E. tarda is also a widespread pathogen which cause infections such as cellulitis or gas gangrene and generalized infections in humans. To control the escalating infection of E. trada on various species, it is essential to decoded the mysterious mechanism behind the bacterial infection at transcript level. In this present study, we carry out a de novo E. tarda Whole transcriptome sequencing, isolated from infected fish intestine using SOLiD sequencing platform. RNA-Seq data analysis was performed using various bioinformatics pipelines. Protein-protein interaction study for pathway enrichment and gene ontology study were executed for further investigation. Assembly statistics for E. tarda dataset showed that the number of transcript contigs was 9657 out of which 6749 were GO annotated whereas 1528 were not assigned any GO terms. GO analysis showed that the expressed genes were enhanced with molecular function, cellular component and biological process. A KEGG enrichment study showed that pathway's that are directly linked with immune diseases like Rheumatoid arthritis (0.2%), Tuberculosis (0.3%) Endocytosis (0.6%) was considerably enriched. Protein-protein interaction study showed that most of the expressed proteins were involved in metabolic pathways, flagellar assembly, Propanoate metabolism, Microbial metabolism in diverse environments, Butanoate metabolism and Carbon. The present study provides novel E. tarda transcriptome sequence data, allowing us to identify biologically significant genes and their functional relationship with fish diseases, and will be useful in recognize the reliable therapeutic targets in near feature.

De novo assembly and functional annotations of the transcriptome of Metorchis orientalis (trematoda: Opisthorchiidae)

Metorchis orientalis is a neglected zoonotic parasite, living in the gallbladder and bile duct of poultry and some mammals as well as humans. In spite of its economic and medical importance, the information known about the transcriptome and genome of M. orientalis is limited. In this study, we performed de novo sequencing, transcriptome assembly and functional annotations of the adult M. orientalis, obtained about 77.4 million high-quality clean reads, among which the length of the transcript contigs ranged from 100 to 11,249 nt with mean length of 373 nt and N50 length of 919 nt. We then assembled 31,943 unigenes, of which 20,009 (62.6%) were annotated by BLASTn and BLASTx searches against the available database. Among these unigenes, 19,795 (62.0%), 3407 (10.7%), 10,620 (33.2%) of them had significant similarity in the NR, NT and Swiss-Prot databases, respectively; 5744 (18.0%) and 4678 (14.6%) unigenes were assigned to GO and COG, respectively; and 9099 (28.5%) unigenes were identified and mapped onto 256 pathways in the KEGG Pathway database. Furthermore, we found that 98 (1.08%) unigenes were related to bile secretion and 5 (0.05%) to primary bile acid biosynthesis pathways category. The characterization of these transcriptomic data has implications for the better understanding of the biology of M. orientalis, and will facilitate the development of intervention agents for this and other pathogenic flukes of human and animal health significance.

De novo assembly of Phlomis purpurea after challenging with Phytophthora cinnamomi

BackgroundPhlomis plants are a source of biological active substances with potential applications in the control of phytopathogens. Phlomis purpurea (Lamiaceae) is autochthonous of southern Iberian Peninsula and Morocco and was found to be resistant to Phytophthora cinnamomi. Phlomis purpurea has revealed antagonistic effect in the rhizosphere of Quercus suber and Q. ilex against P. cinnamomi. Phlomis purpurea roots produce bioactive compounds exhibiting antitumor and anti-Phytophthora activities with potential to protect susceptible plants. Although these important capacities of P. purpurea have been demonstrated, there is no transcriptomic or genomic information available in public databases that could bring insights on the genes underlying this anti-oomycete activity.ResultsUsing Illumina technology we obtained a de novo assembly of P. purpurea transcriptome and differential transcript abundance to identify putative defence related genes in challenged versus non-challenged plants. A total of 1,272,600,000 reads from 18 cDNA libraries were merged and assembled into 215,739 transcript contigs. BLASTX alignment to Nr NCBI database identified 124,386 unique annotated transcripts (57.7%) with significant hits. Functional annotation identified 83,550 out of 124,386 unique transcripts, which were mapped to 141 pathways.39% of unigenes were assigned GO terms. Their functions cover biological processes, cellular component and molecular functions. Genes associated with response to stimuli, cellular and primary metabolic processes, catalytic and transporter functions were among those identified.Differential transcript abundance analysis using DESeq revealed significant differences among libraries depending on post-challenge times.Comparative cyto-histological studies of P. purpurea roots challenged with P. cinnamomi zoospores and controls revealed specific morphological features (exodermal strips and epi-cuticular layer), that may provide a constitutive efficient barrier against pathogen penetration. Genes involved in cutin biosynthesis and in exodermal Casparian strips formation were up-regulated.ConclusionsThe de novo assembly of transcriptome using short reads for a non-model plant, P. purpurea, revealed many unique transcripts useful for further gene expression, biological function, genomics and functional genomics studies.The data presented suggest a combination of a constitutive resistance and an increased transcriptional response from P. purpurea when challenged with the pathogen. This knowledge opens new perspectives for the understanding of defence responses underlying pathogenic oomycete/plant interaction upon challenge with P. cinnamomi.

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing

BackgroundDespite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms.ResultsThe sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes.ConclusionsThe transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.

Improving de novo metatranscriptome assembly via machine learning algorithms

In this paper, we present DNPipe, a Pipeline that processes and filters the transcript contigs reported by existing De Novo metatranscriptome assembly algorithms, aiming to improve the quality of de novo assembly of metatranscriptomic sequences. DNPipe consists of expectation-maximisation (EM) and sampling approaches that utilise abundance information of transcript contigs. We tested DNPipe on six metatranscriptomic datasets acquired from a mock microbial community dominated by three (among more than 15) known bacterial genomes. Results show that DNPipe can substantially improve the quality of metatranscriptome assembly, producing longer and more accurate transcripts. The N50 of the contigs increases by 19% (from around 1880 bps to 2250 bps), and the precision of the assembly improves by up to 8.7%, achieving about 81%. DNPipe assemblies are of higher quality than those assembled by Trinity as well. The DNPipe tool can be downloaded as open source software at https://sourceforge.net/projects/dnpipe/files/.

Improving de novo metatranscriptome assembly via machine learning algorithms

In this paper, we present DNPipe, a Pipeline that processes and filters the transcript contigs reported by existing De Novo metatranscriptome assembly algorithms, aiming to improve the quality of de novo assembly of metatranscriptomic sequences. DNPipe consists of expectation-maximisation (EM) and sampling approaches that utilise abundance information of transcript contigs. We tested DNPipe on six metatranscriptomic datasets acquired from a mock microbial community dominated by three (among more than 15) known bacterial genomes. Results show that DNPipe can substantially improve the quality of metatranscriptome assembly, producing longer and more accurate transcripts. The N50 of the contigs increases by 19% (from around 1880 bps to 2250 bps), and the precision of the assembly improves by up to 8.7%, achieving about 81%. DNPipe assemblies are of higher quality than those assembled by Trinity as well. The DNPipe tool can be downloaded as open source software at https://sourceforge.net/projects/dnpipe/files/.

Doubled Haploid 'CUDH2107' as a Reference for Bulb Onion (Allium cepa L.) Research: Development of a Transcriptome Catalogue and Identification of Transcripts Associated with Male Fertility.

Researchers working on model plants have derived great benefit from developing genomic and genetic resources using ‘reference’ genotypes. Onion has a large and highly heterozygous genome making the sharing of germplasm and analysis of sequencing data complicated. To simplify the discovery and analysis of genes underlying important onion traits, we are promoting the use of the homozygous double haploid line ‘CUDH2107’ by the onion research community. In the present investigation, we performed transcriptome sequencing on vegetative and reproductive tissues of CUDH2107 to develop a multi-organ reference transcriptome catalogue. A total of 396 million 100 base pair paired reads was assembled using the Trinity pipeline, resulting in 271,665 transcript contigs. This dataset was analysed for gene ontology and transcripts were classified on the basis of putative biological processes, molecular function and cellular localization. Significant differences were observed in transcript expression profiles between different tissues. To demonstrate the utility of our CUDH2107 transcriptome catalogue for understanding the genetic and molecular basis of various traits, we identified orthologues of rice genes involved in male fertility and flower development. These genes provide an excellent starting point for studying the molecular regulation, and the engineering of reproductive traits.

Transcriptome analysis in the beet webworm, Spoladea recurvalis (Lepidoptera: Crambidae).

The beet webworm, Spoladea recurvalis Fabricius, is a destructive pest on vegetable crops in tropics and subtropics; its main host plant is amaranth. It has become imperative to develop non-chemical methods to control S. recurvalis on amaranth. However, the lack of molecular information about this species has hindered the development of novel pest management strategies. In this study, high-throughput RNA sequencing covering de novo sequence assemblies, functional annotation of transcripts, gene function classification and enrichment was performed on S. recurvalis. Illumina sequencing generated a total of 120435 transcript contigs ranging from 201 to 22729 bases with a mean length of 688 bases. The assembled transcripts were subjected to Basic Local Alignment Search Tool-X (BLASTX) to obtain the annotations against non-redundant, Swiss-Prot, Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases. A subset of 58225 transcript sequences returned hits from known proteins in the National Center for Biotechnology Information database, and the majority of the transcript sequences had the highest number of hits for Danaus plexippus (50.43%). A total of 1217 Gene Ontology-level 3 annotations were assigned to 51805 transcripts, while 39650 transcripts were predicted as functional protein-coding genes in the COG database and 20037 transcripts were enriched to KEGG pathways. We identified 40 putative genes related to pheromone production and reception in S. recurvalis, with the expression of one gene between 0.29 and 1141.79 fragments per kilo base per million (FPKM) reads. The transcriptome sequence of S. recurvalis is a first step toward offering a comprehensive genomic resource which would enable better understanding of molecular mechanisms to enable development of effective pest management practices for this species.

RNA Sequencing Revealed Numerous Polyketide Synthase Genes in the Harmful Dinoflagellate Karenia mikimotoi.

The dinoflagellate Karenia mikimotoi forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of K. mikimotoi on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of K. mikimotoi. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS) sequences. Thus, K. mikimotoi was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of Karenia brevis. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight Karenia PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by Karenia species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (sxtA) in the toxic dinoflagellate Alexandrium fundyense. These contigs encoded A1–A3 domains of sxtA genes. Thus, this study identified some transcripts in K. mikimotoi that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of K. mikimotoi and other dinoflagellates.

De novo assembly and characterization of the transcriptome of the pancreatic fluke Eurytrema pancreaticum (trematoda: Dicrocoeliidae) using Illumina paired-end sequencing

Eurytrema pancreaticum is one of the most common trematodes living in the pancreatic and bile ducts of ruminants and also occasionally infects humans, causing eurytremiasis. In spite of its economic and medical importance, very little is known about the genomic resources of this parasite. Herein, we performed de novo sequencing, assembly and characterization of the transcriptome of adult E. pancreaticum. Approximately 36.4 million high-quality clean reads were obtained, and the length of the transcript contigs ranged from 66 to 19,968nt with mean length of 479nt and N50 length of 1094nt, and then 23,573 unigenes were assembled. Of these unigenes, 15,353 (65.1%) were annotated by blast searches against the NCBI non-redundant protein database. Among these, 15,267 (64.8%), 2732 (11.6%) and 10,354 (43.9%) of the unigenes had significant similarity with proteins in the NR, NT and Swiss-Prot databases, respectively. 5510 (23.4%) and 4567 (19.4%) unigenes were assigned to GO and COG, respectively. 8886 (37.7%) unigenes were identified and mapped onto 254 pathways in the KEGG Pathway database. Furthermore, we found that 105 (1.18%) unigenes were related to pancreatic secretion and 61 (0.7%) to pancreatic cancer. The present study represents the first transcriptome of any members of the family Dicrocoeliidae, which has little genomic information available in the public databases. The novel transcriptome of E. pancreaticum should provide a useful resource for designing new strategies against pancreatic flukes and other trematodes of human and animal health significance.

UNRAVELING THE MOLECULAR INTERACTION BETWEEN PSEUDOMONAS SYRINGAE PV. ACTINIDIAE (PSA) AND THE KIWIFRUIT PLANT THROUGH RNASEQ APPROACH

Bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is the most important disease of kiwifruit, threatening kiwifruit production around the world. However, little is known about the molecular interaction between Psa and kiwifruit plants. To elucidate early molecular plant response during the different phases of the infection, gene expression in healthy or inoculated plants of Actinidia chinensis was analyzed 3, 24 and 48 h after experimental inoculation. In addition, to understand the molecular mechanism underlying the increased plant resistance observed after the exogenous application of the systemic acquired resistance (SAR) inducer acibenzolar- S-methyl (ASM), gene expression was analyzed in healthy or inoculated ASM pre-treated plants. Transcriptome analysis of A. chinensis, was performed using an Illumina GAIIx sequencer, generating 221 million 75 bp long paired end reads. The raw reads were de novo assembled into 63,943 contigs by Trinity software with an average size of 1,078 bp and N50 of 1,614 bp. Annotation of the de novo transcript contigs was done using Blast2GO software, BLASTX was performed against the NCBI non-redundant protein database with an E-value cut-off of 1e-5. Mapped records were also aligned to protein databases such as Swiss-Prot, KEGG and COG, in order to retrieve proteins with the highest sequence similarity, to give a putative functional annotation. Finally, InterProScan was used to identify additional gene ontology (GO) annotations. Reads were mapped to the contigs using the CLC software to identify differentially expressed genes (DEGs), statistical analysis was done using the method described in the R package DESeq. A preliminary analysis of DEGs was conducted on sequences obtained 24 h after infection. A total of 349 DEGs were identified in response to infection in non-ASM pre-treated plants. When ASM pre-treated plants were considered, 637 DEGs were identified, 213 of them were in common with the 349 DEGs modulated by infection in not ASM pre-treated plants. The ASM treatment by itself was able to modulated 974 DEGs, 70 of them were further modulated by infection in ASM pre-treated plants. These results suggest that the molecular response to Psa is strongly enhanced in ASM pre-treated plants.

De novo Assembly, Characterization of Immature Seed Transcriptome and Development of Genic-SSR Markers in Black Gram [Vigna mungo (L.) Hepper

Black gram [V. mungo (L.) Hepper] is an important legume crop extensively grown in south and south-east Asia, where it is a major source of dietary protein for its predominantly vegetarian population. However, lack of genomic information and markers has become a limitation for genetic improvement of this crop. Here, we report the transcriptome sequencing of the immature seeds of black gram cv. TU94-2, by Illumina paired end sequencing technology to generate transcriptome sequences for gene discovery and genic-SSR marker development. A total of 17.2 million paired-end reads were generated and 48,291 transcript contigs (TCS) were assembled with an average length of 443 bp. Based on sequence similarity search, 33,766 TCS showed significant similarity to known proteins. Among these, only 29,564 TCS were annotated with gene ontology (GO) functional categories. A total number of 138 unique KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified, of which majority of TCS are grouped into purine metabolism (678) followed by pyrimidine metabolism (263). A total of 48,291 TCS were searched for SSRs and 1,840 SSRs were identified in 1,572 TCS with an average frequency of one SSR per 11.9 kb. The tri-nucleotide repeats were most abundant (35%) followed by di-nucleotide repeats (32%). PCR primer pairs were successfully designed for 933 SSR loci. Sequences analyses indicate that about 64.4% and 35.6% of the SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Tri-nucleotide repeats (57.3%) were preferentially present in the CDS. The rate of successful amplification and polymorphism were investigated using selected primers among 18 black gram accessions. Genic-SSR markers developed from the Illumina paired end sequencing of black gram immature seed transcriptome will provide a valuable resource for genetic diversity, evolution, linkage mapping, comparative genomics and marker-assisted selection in black gram.

Transcriptome analysis of the adult rumen fluke Paramphistomum cervi following next generation sequencing

Rumen flukes are parasitic trematodes (Platyhelminthes: Digenea) of major socioeconomic importance in many countries. Key representatives, such as Paramphistomum cervi, can cause “Rumen fluke disease” or paramphistomosis and undermine economic animal productivity and welfare. P. cervi is primarily a problem in sheep, goat and buffalo production as a consequence of reduced weight gain and milk production, clinical disease or death. Recent technological advances in genomics and bioinformatics now provide unique opportunities for the identification and pre-validation of drug targets and vaccines through improved understanding of the biology of pathogens such as P. cervi and their relationship with their hosts at the molecular level. Here, we report next generation transcriptome sequencing analysis for P. cervi. RNAseq libraries were generated from RNA extracted from 15 adult P. cervi parasites sampled from each of three different host species (sheep, goat and buffalo) and a reference transcriptome was generated by assembly of all Ion Torrent PGM sequencing data. Raw reads (7,433,721 in total) were initially filtered for host nucleotide contamination and ribosomal RNAs and the remaining reads were assembled into 43,753 high confidence transcript contigs. In excess of 50% of the assembled transcripts were annotated with domain- or protein sequence similarity derived functional information. The reference adult P. cervi transcriptome will serve as a basis for future work on the biology of this important parasite. Using the widely investigated trematode virulence factor and vaccine candidate Cathepsin L as an example, the epitope GPISIAINA was found to be conserved in P. cervi isolated from three different host species supporting its candidacy for vaccine development and illustrating the utility of the adult P. cervi transcriptome.

Genome-wide transcriptomic and phylogenetic analyses reveal distinct aluminum-tolerance mechanisms in the aluminum-accumulating species buckwheat (Fagopyrum tataricum).

BackgroundSimilar to common buckwheat (Fagopyrum esculentum), tartary buckwheat (Fagopyrum tataricum) shows a high level of aluminum (Al) tolerance and accumulation. However, the molecular mechanisms for Al detoxification and accumulation are still poorly understood. To begin to elucidate the molecular basis of Al tolerance and accumulation, we used the Illumina high-throughput mRNA sequencing (RNA-seq) technology to conduct a genome-wide transcriptome analysis on both tip and basal segments of the roots exposed to Al.ResultsBy using the Trinity method for the de novo assembly and cap3 software to reduce the redundancy and chimeras of the transcripts, we constructed 39,815 transcripts with an average length of 1184 bp, among which 20,605 transcripts were annotated by BLAST searches in the NCBI non-redundant protein database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that expression of genes involved in the defense of cell wall toxicity and oxidative stress was preferentially induced by Al stress. Our RNA-seq data also revealed that organic acid metabolism was unlikely to be a rate-limiting step for the Al-induced secretion of organic acids in buckwheat. We identified two citrate transporter genes that were highly induced by Al and potentially involved in the release of citrate into the xylem. In addition, three of four conserved Al-tolerance genes were found to be duplicated in tartary buckwheat and display diverse expression patterns.ConclusionsNearly 40,000 high quality transcript contigs were de novo assembled for tartary buckwheat, providing a reference platform for future research work in this plant species. Our differential expression and phylogenetic analysis revealed novel aspects of Al-tolerant mechanisms in buckwheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0395-z) contains supplementary material, which is available to authorized users.

Transcriptome of Teak (Tectona grandis, L.f) in Vegetative to Generative Stages Development

Teak is one of the highly famous woody plant species for its premier quality of wood. Teak has problem on productivity because of long reproductive cycle. The problem is basically related to mechanism of flower development. The aim of this study was preliminary development of expressed gene database to characterize the floral transcriptome in teak. Two subtracted cDNA libraries were constructed from vegetative and generative bud tissues. Libraries were sequenced using Illumina MiSeq technology which generated paired-end read sequences 3,778,316 for vegetative and 3,701,878 for generative. The sequences assembled de novo into 87,365 transcript contigs consisting of 42,435,728 bases with N50 of 498 bp using CLC-Genomics Workbench. 76,169 (87.18%) of the 87,365 assembled contigs exhibited significant similarity BLASTN to Solanum lycopersicum database (www.phytozome.com). The assembled contigs were annotated through high stringency BLASTX analysis to proteome of S. lycopersicum. Distribution of contigs abundance between vegetative and generative stages analyzed using the DEGseq approach. The numbers of contigs distribution are 24,730 in vegetative, 28,912 in generative and 33,723 in both stages. The functionally protein datasets characterized by Gene Ontology (GO) annotation and KEGG metabolic pathways assignments for the result of DEG analysis. These contigs, 18,756 (75.84%) from vegetative, 22,089 (76.40%) from generative and 22,917 (67.96%) from both stages were assigned to GO classes. A total of 1455 (13.77%) were mapped to 30 pathways from vegetative, 1,638 (13.70%) were mapped to 27 pathways from generative and 1,652 (12.20%) were mapped to 30 pathways from both by BLAST comparison against the KEGG database. The biological processes of flowering developments were identified in the biological process dataset and the numbers of contigs were discovered different between stages. This transcriptome dataset information will act as a valuable resource for further molecular genetic studies teak, as well as for isolation and characterization of functional genes involved in flowering development pathways.