De Novo Transcriptome Sequencing Research Articles

De novo sequencing and transcriptome analysis of Indian Bael (Aegle marmelos L.)

Aegle marmelos (L.) also known as Bael is a medicinally important tree known for its antidiabetic properties. It is considered as a sacred plant but lacks sufficient genomic and transcriptomic resources. The present study investigated functional profile of A. marmelos via RNA sequencing technology. The denovo transcriptome sequencing and assembly of 4.7 million high quality reads yielded 27,393 unigenes, which were annotated against the databases such as NCBI (National Centre for Biotechnology Information), NR (Non redundant) database, KEGG, Pfam (Protein Family), CDD (Conserved Domains database) and Uniprot. The annotation of assembled transcripts showed a total of 1291 transcripts mapped to 81 different transcription factor families, whereas 55 transcripts were assigned to CYP450 belonging to 21 different classes. KEGG mapping showed transcripts involved in the biosynthesis of terpenoids, flavonoids, including transcripts for synthesis of important compounds like Vitamin E, 4-coumarate and Vitamin K epoxide. 1211 simple sequence repeats (SSRs) were identified which may aid in molecular breeding studies. The study provides an insight into the expression profile of Bael and an opportunity for future genomic and metabolic studies in A.marmelos.

De Novo transcriptome sequencing explored cultivar specific sequence variation and differential expression of pigment synthesis genes in turmeric (Curcuma longa L.)

Turmeric contains a yellow color pigment that is curcumin, based on which different cultivars of turmeric have been released so far. Limited molecular studies on different pigment synthesis genes and lack of cultivar specific SSRs and SNPs in turmeric have confined the scope of identification of turmeric and establishment of their genetic makeup. In this study de novo transcriptome assembly of four turmeric cultivars i.e. Suroma, Roma, Lakadong and Megha was done. From the transcriptome analysis, cultivar specific SSRs, SNPs and transcripts encoding pigment synthesis genes were derived. A total of 113,862 contigs for Suroma, 125,762 for Roma, 113,021 for Lakadong and 116,711 for Megha were obtained. Multiple databases were used to acquire a complete annotation and the transcripts were functionally categorized using GO, KOG, KEGG and PlantCyc database. While presenting a large repertoire of cultivar specific quality SNPs and variations in SSRs, we have also demonstrated the functional basis of differentially expressed pigment synthesis genes among the four cultivars. The results of this study have clearly indicated their different genetic makeup. This report would enable authentic identification of these morphologically similar elite cultivars through use of cultivar specific SSRs and SNPs and to understand the molecular mechanisms behind differential expression of specific pigment synthesis genes in turmeric while providing scope for overall improvement of the taxa.

De Novo Transcriptome Sequencing Analysis of Goose (Anser anser) Embryonic Skin and the Identification of Genes Related to Feather Follicle Morphogenesis at Three Stages of Development.

The objective of this study was to evaluate the changes in the goose embryo transcriptome during feather development. RNA-Sequencing (RNA-Seq) was used to find the transcriptome profiles of feather follicles from three stages of embryonic dorsal skin at embryonic day 13, 18, and 28 (E13, E18, E28). The results showed that 3001, 6634, and 13,780 genes were differently expressed in three stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that differentially expressed genes (DEGs) in E13 vs. E18 were significantly mapped into the GO term of extracellular structure organization and the pathway of extracellular matrix (ECM)-receptor interaction. In E18 vs. E28, the top significantly mapped into GO term was the single-organism developmental process; the pathway was also the ECM-receptor interaction. DEGs in E13 vs. E28 were significantly mapped into the GO term of the multicellular organismal process and the pathway of cell adhesion molecules. Subsequently, the union of DEGs was categorized by succession cluster into eight profiles, which were then grouped into four ideal profiles. Lastly, the seven genes spatio-temporal expression pattern was confirmed by real-time PCR. Our findings advocate that interleukin 20 receptor subunit alpha (IL20RA), interleukin 6 receptor (IL6R), interleukin 1 receptor type 1 (IL-1R1), Wnt family member 3A (WNT3A), insulin-like growth factor binding protein 3 (IGFBP3), bone morphogenetic protein 7 (BMP7), and secreted-frizzled related protein 2 (SFRP2) might possibly play vital roles in skin and feather follicle development and growth processes.

De Novo Transcriptome Sequencing and Analysis to Identify Genes Involved in the Biosynthesis of Flavonoids in Abrus mollis Leaves

Abrus mollis Hance is an endemic genuine medicinal material growing widely in Southern China. We used the next-generation sequencing technology to carry out de novo transcriptome sequencing and analysis of A. mollis leaves. 57669118 high quality reads and 53743 assembled unigenes were obtained. 36271 unigenes (67.49%) were annotated in the four public databases (NCBI-nr, Swiss-Prot, KEGG, and KOG). After comprehensive analysis of the A. mollis transcriptome, 35656 unigenes were classified into 25 KOG function classifications and 7572 unigenes were assigned to 128 different KEGG pathways. Then, the unigenes with their key enzymes in phenylalanine, phenylpropanoid, flavonoids and isoflavonoid biosynthesis pathway were investigated in KEGG pathways. The study results will be helpful to reveal the biosynthesis mechanism of flavonoids and isoflavonoid in A. mollis leaves and carry out its biological regulation research.

De Novo Transcriptome Sequencing and the Hypothetical Cold Response Mode of Saussurea involucrata in Extreme Cold Environments.

Saussurea involucrata grows in high mountain areas covered by snow throughout the year. The temperature of this habitat can change drastically in one day. To gain a better understanding of the cold response signaling pathways and molecular metabolic reactions involved in cold stress tolerance, genome-wide transcriptional analyses were performed using RNA-Seq technologies. A total of 199,758 transcripts were assembled, producing 138,540 unigenes with 46.8 Gb clean data. Overall, 184,416 (92.32%) transcripts were successfully annotated. The 365 transcription factors identified (292 unigenes) belonged to 49 transcription factor families associated with cold stress responses. A total of 343 transcripts on the signal transduction (132 upregulated and 212 downregulated in at least any one of the conditions) were strongly affected by cold temperature, such as the CBL-interacting serine/threonine-protein kinase (CIPKs), receptor-like protein kinases, and protein kinases. The circadian rhythm pathway was activated by cold adaptation, which was necessary to endure the severe temperature changes within a day. There were 346 differentially expressed genes (DEGs) related to transport, of which 138 were upregulated and 22 were downregulated in at least any one of the conditions. Under cold stress conditions, transcriptional regulation, molecular transport, and signal transduction were involved in the adaptation to low temperature in S. involucrata. These findings contribute to our understanding of the adaptation of plants to harsh environments and the survival traits of S. involucrata. In addition, the present study provides insight into the molecular mechanisms of chilling and freezing tolerance.

Transcriptome analysis of colored calla lily (Zantedeschia rehmanniiEngl.) by Illumina sequencing:de novoassembly, annotation and EST-SSR marker development

Colored calla lily is the short name for the species or hybrids in section Aestivae of genus Zantedeschia. It is currently one of the most popular flower plants in the world due to its beautiful flower spathe and long postharvest life. However, little genomic information and few molecular markers are available for its genetic improvement. Here, de novo transcriptome sequencing was performed to produce large transcript sequences for Z. rehmannii cv. ‘Rehmannii’ using an Illumina HiSeq 2000 instrument. More than 59.9 million cDNA sequence reads were obtained and assembled into 39,298 unigenes with an average length of 1,038 bp. Among these, 21,077 unigenes showed significant similarity to protein sequences in the non-redundant protein database (Nr) and in the Swiss-Prot, Gene Ontology (GO), Cluster of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, a total of 117 unique transcripts were then defined that might regulate the flower spathe development of colored calla lily. Additionally, 9,933 simple sequence repeats (SSRs) and 7,162 single nucleotide polymorphisms (SNPs) were identified as putative molecular markers. High-quality primers for 200 SSR loci were designed and selected, of which 58 amplified reproducible amplicons were polymorphic among 21 accessions of colored calla lily. The sequence information and molecular markers in the present study will provide valuable resources for genetic diversity analysis, germplasm characterization and marker-assisted selection in the genus Zantedeschia.

De Novo Transcriptome Sequencing of Low Temperature-Treated Phlox subulata and Analysis of the Genes Involved in Cold Stress.

Phlox subulata, a perennial herbaceous flower, can survive during the winter of northeast China, where the temperature can drop to −30 °C, suggesting that P. subulata is an ideal model for studying the molecular mechanisms of cold acclimation in plants. However, little is known about the gene expression profile of P. subulata under cold stress. Here, we examined changes in cold stress-related genes in P. subulata. We sequenced three cold-treated (CT) and control (CK) samples of P. subulata. After de novo assembly and quantitative assessment of the obtained reads, 99,174 unigenes were generated. Based on similarity searches with known proteins in public protein databases, 59,994 unigenes were functionally annotated. Among all differentially expressed genes (DEGs), 8302, 10,638 and 11,021 up-regulated genes and 9898, 17,876, and 12,358 down-regulated genes were identified after treatment at 4, 0, and −10 °C, respectively. Furthermore, 3417 up-regulated unigenes were expressed only in CT samples. Twenty major cold-related genes, including transcription factors, antioxidant enzymes, osmoregulation proteins, and Ca2+ and ABA signaling components, were identified, and their expression levels were estimated. Overall, this is the first transcriptome sequencing of this plant species under cold stress. Studies of DEGs involved in cold-related metabolic pathways may facilitate the discovery of cold-resistance genes.

Plant Comparative and Functional Genomics.

Plant Comparative and Functional Genomics.

Characterization and expression of sweetfish (Pleco glossus altivelis) cathepsin D.

Cathepsin D (CTSD) is a lysosomal acidic endoproteinase that plays an important role in immune response. In this study, we obtained sweetfish (Plecoglossus altivelis) CTSD (PaCTSD) via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1 955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17 × 10³. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.

Microsatellites for the marsh fritillary butterfly: de novo transcriptome sequencing, and a comparison with amplified fragment length polymorphism (AFLP) markers.

BackgroundUntil recently the isolation of microsatellite markers from Lepidoptera has proved troublesome, expensive and time-consuming. Following on from a previous study of Edith's checkerspot butterfly, Euphydryas editha, we developed novel microsatellite markers for the vulnerable marsh fritillary butterfly, E. aurinia. Our goal was to optimize the process in order to reduce both time and cost relative to prevailing techniques. This was accomplished by using a combination of previously developed techniques: in silico mining of a de novo assembled transcriptome sequence, and genotyping the microsatellites found there using an economic method of fluorescently labelling primers.Principal FindingsIn total, we screened nine polymorphic microsatellite markers, two of which were previously published, and seven that were isolated de novo. These markers were able to amplify across geographically isolated populations throughout Continental Europe and the UK. Significant deviations from Hardy-Weinberg equilibrium were evident in some populations, most likely due to the presence of null alleles. However, we used an Fst outlier approach to show that these markers are likely selectively neutral. Furthermore, using a set of 128 individuals from 11 populations, we demonstrate consistency in population differentiation estimates with previously developed amplified fragment length polymorphism (AFLP) markers (r = 0.68, p<0.001).SignificanceRapid development of microsatellite markers for difficult taxa such as Lepidoptera, and concordant results with other putatively neutral molecular markers, demonstrate the potential of de novo transcriptional sequencing for future studies of population structure and gene flow that are desperately needed for declining species across fragmented landscapes.

De-Novo Transcriptome Sequencing of a Normalized cDNA Pool from Influenza Infected Ferrets

The ferret is commonly used as a model for studies of infectious diseases. The genomic sequence of this animal model is not yet characterized, and only a limited number of fully annotated cDNAs are currently available in GenBank. The majority of genes involved in innate or adaptive immune response are still lacking, restricting molecular genetic analysis of host response in the ferret model. To enable de novo identification of transcriptionally active ferret genes in response to infection, we performed de-novo transcriptome sequencing of animals infected with H1N1 A/California/07/2009. We also included splenocytes induced with bacterial lipopolysaccharide to allow for identification of transcripts specifically induced by Gram-negative bacteria. We pooled and normalized the cDNA library in order to delimit the risk of sequencing only highly expressed genes. While normalization of the cDNA library removes the possibility of assessing expression changes between individual animals, it has been shown to increase identification of low abundant transcripts. In this study, we identified more than 19000 partial ferret transcripts, including more than 1000 gene orthologs known to be involved in the innate and the adaptive immune response.

Rapid Microsatellite Isolation from a Butterfly by De Novo Transcriptome Sequencing: Performance and a Comparison with AFLP-Derived Distances

BackgroundThe isolation of microsatellite markers remains laborious and expensive. For some taxa, such as Lepidoptera, development of microsatellite markers has been particularly difficult, as many markers appear to be located in repetitive DNA and have nearly identical flanking regions. We attempted to circumvent this problem by bioinformatic mining of microsatellite sequences from a de novo-sequenced transcriptome of a butterfly (Euphydryas editha).Principal FindingsBy searching the assembled sequence data for perfect microsatellite repeats we found 10 polymorphic loci. Although, like many expressed sequence tag-derived microsatellites, our markers show strong deviations from Hardy-Weinberg equilibrium in many populations, and, in some cases, a high incidence of null alleles, we show that they nonetheless provide measures of population differentiation consistent with those obtained by amplified fragment length polymorphism analysis. Estimates of pairwise population differentiation between 23 populations were concordant between microsatellite-derived data and AFLP analysis of the same samples (r = 0.71, p<0.00001, 425 individuals from 23 populations).Significance De novo transcriptional sequencing appears to be a rapid and cost-effective tool for developing microsatellite markers for difficult genomes.