Full-length Non-chimeric Reads Research Articles

Full-length transcriptome analysis of a bloom-forming dinoflagellate Prorocentrum shikokuense (Dinophyceae)

Prorocentrum shikokuense (formerly P. donghaiense) is a pivotal dinoflagellate species associating with the HABs in the East China Sea. The complexity of its large nuclear genome hindered us from understanding its genomic characteristics. Full-length transcriptome sequencing offers a practical solution to decipher the physiological mechanisms of a species without the reference genome. In this study, we employed single-molecule real-time (SMRT) sequencing technology to sequence the full-length transcriptome of Prorocentrum shikokuense. We successfully generated 41.73 Gb of clean SMRT sequencing reads and isolated 105,249 non-redundant full-length non-chimeric reads. Our trial has led to the identification of 11,917 long non-coding RNA transcripts, 514 alternative splicing events, 437 putative transcription factor genes from 17 TF gene families, and 34,723 simple sequence repeats. Additionally, a total of 78,265 open reading frames were identified, of them 15,501 were the protein coding sequences. This dataset is valuable for annotating P. shikokuense genome, and will contribute significantly to the in-depth studies on the molecular mechanisms underlining the dinoflagellate bloom formation.

Comprehensive Profiling of Alternative Splicing and Alternative Polyadenylation during Fruit Ripening in Watermelon (Citrullus lanatus).

Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening.

The sequencing of the key genes and end products in the TLR4 signaling pathway from the kidney of Rana dybowskii exposed to Aeromonas hydrophila.

Infectious diseases caused by Aeromonas hydrophila (AH) have reduced the populations of Rana dybowskii). However, little is known about the immune response of R. dybowskii against AH infections. The toll-like receptor (TLR) signaling pathway has been identified as a critical component in innate immunity, responsible for identifying pathogen-associated molecular patterns in pathogens. Our study used the next-generation sequencing technique and single-molecule long-read sequencing to determine the structures of transcript isoforms and functions of genes in the kidneys of R. dybowskii, as well as identify and validate the related genes in the TLR4 signaling pathway. In total, 628,774 reads of inserts were identified, including 300,053 full-length non-chimeric reads and 233,592 non-full-length reads. Among the transcriptome sequences, 124 genes were identified as homologs of known genes in the TLR4 pathway especially inflammatory cytokines and receptors. Our findings shed light on the structures and functions of R. dybowskii genes exposed to AH and confirm the presence of both MyD88-dependent and independent pathways in R. dybowskii. Our work reveals how various functional proteins in amphibians at the initial stage of immune response are activated and complete their corresponding functions in a short time.

De novo high-accuracy transcriptomes from long-read sequencing reveals a wide variety of novel splice variants in copepodids and adult female salmon lice (Lepeophtheirus salmonis)

Former transcriptome studies of the ectoparasitic salmon louse (Lepeophtheirus salmonis) are based on short-read sequencing and in silico predictions, with the disadvantage of inadequately describing splice variants and insufficient differentiation between duplicated genes. In the present study, a de novo full-length transcriptome (TSA accession GKKU00000000) was generated using single-molecule long-read RNA-sequencing (PacBio IsoSeq platform) corrected by short reads (Illumina platform) from the same RNA samples. The two samples, cephalothorax of an adult female and her copepodid offspring, were analyzed separately to facilitate comparison and identification of transcripts unique to each life stage. Each transcript has been supported by two or more full-length non-chimeric reads and at least three short reads, ensuring high-sequence accuracy. A total of 31,092 unique high-accuracy full-length transcripts with an open reading frame > 150 bp, originating from 10,034 unique loci of the salmon louse genome, were identified. More than half of the transcripts are life-stage specific, exclusively present in either the copepodid or adult sample. Approximately one-third of the transcripts were full splice matches with predicted protein coding transcripts presented in NCBI, thus validating these. More than half of the transcripts constituted novel isoforms with at least one new splicing site. We conclude that the full-length transcriptomes represent a versatile reference resource of transcripts. Suitable applications include expression studies, SNP mining, and studies on the biological effects of differences in gene (or isoform) expression between copepodids and adult females. The additional functional annotation of 88% of transcripts allows for identification of gene families of particular interest and for exploration of gene networks and enrichment analysis following expression studies.

A survey of the full-length transcriptome of Gracilariopsis lemaneiformis using single-molecule long-read sequencing

BackgroundPosttranscriptional processing of precursor mRNAs contributes to transcriptome and protein diversity and gene regulatory mechanisms in eukaryotes. However, this posttranscriptional mechanism has not been studied in the marine macroalgae Gracilariopsis lemaneiformis, which is the most cultivated red seaweed species in China.ResultsIn the present study, third-generation sequencing (Pacific Biosciences single-molecule real-time long-read sequencing, SMRT-Seq) was used to sequence the full-length transcriptome of G. lemaneiformis to identify alternatively spliced transcripts and alternative polyadenylation (APA) sites in this species. RNAs were isolated from G. lemaneiformis under various treatments including abiotic stresses and exogenous phytohormones, and then equally pooled for SMRT-Seq. In summary, 346,544 full-length nonchimeric reads were generated, from which 13,630 unique full-length transcripts were obtained in G. lemaneiformis. Compared with the known splicing events in the gene models, more than 3000 new alternative splicing (AS) events were identified in the SMRT-Seq reads. Additionally, 810 genes were found to have poly (A) sites and 91 microRNAs (miRNAs), 961 long noncoding RNAs and 1721 novel genes were identified in G. lemaneiformis. Moreover, validation experiments showed that abiotic stresses and phytohormones could induce some specific AS events, especially intron retain isoforms, cause some alterations to the relative ratios of transcripts annotated to the same gene, and generate novel 3′ ends because of differential APA. The growth of G. lemaneiformis was inhibited by Cu stress, while this inhibition was alleviated by ACC treatment. RNA-Seq analysis further revealed that 211 differential alternative splicing (DAS) events and 142 DAS events was obtained in CK vs Cu and Cu vs Cu + ACC, respectively, suggesting that AS of functional genes could be regulated by Cu stress and ACC. Compared with Cu stress, the expression of transcripts with DAS events mainly involved in the carbon fixation in photosynthetic organisms and oxidative phosphorylation pathway was upregulated in Cu + ACC treatment, revealing that ACC alleviated the growth inhibition by Cu stress by increasing carbon fixation and oxidative phosphorylation.ConclusionsOur results provide the first comprehensive picture of the full-length transcriptome and posttranscriptional mechanism in red macroalgae, including transcripts that appeared in the presence of common abiotic stresses and phytohormones, which will improve the gene annotations of Gracilariopsis and contribute to the study of gene regulation in this important cultivated seaweed.

RNA-Seq and Iso-Seq Reveal the Important Role of COMT and CCoAOMT Genes in Accumulation of Scopoletin in Noni (Morinda citrifolia).

Scopoletin, the main component of clinical drugs and the functional component of health products, is highly abundant in noni fruit (Morinda citrifolia). Multiple enzyme genes regulate scopoletin accumulation. In the present study, differentially expressed genes of noni were analyzed by RNA sequencing (RNA-Seq) and the full-length genes by isoform-sequencing (Iso-Seq) to find the critical genes in the scopoletin accumulation mechanism pathway. A total of 32,682 full-length nonchimeric reads (FLNC) were obtained, out of which 16,620 non-redundant transcripts were validated. Based on KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation and differential expression analysis, two differentially expressed genes, caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA O-methyltransferase (CCoAOMT), were found in the scopoletin accumulation pathway of noni. Real-time quantitative polymerase chain reaction (q-PCR), phylogenetic tree analysis, gene expression analysis, and the change in scopoletin content confirmed that these two proteins are important in this pathway. Based on these results, the current study supposed that COMT and CCoAOMT play a significant role in the accumulation of scopoletin in noni fruit, and COMT (gene number: gene 7446, gene 8422, and gene 6794) and CCoAOMT (gene number: gene 12,084) were more significant. These results provide the importance of COMT and CCoAOMT and a basis for further understanding the accumulation mechanism of scopoletin in noni.

Full-length transcriptome analysis of the bloom-forming dinoflagellate Akashiwo sanguinea by single-molecule real-time sequencing.

The dinoflagellate Akashiwo sanguinea is a harmful algal species and commonly observed in estuarine and coastal waters around the world. Harmful algal blooms (HABs) caused by this species lead to serious environmental impacts in the coastal waters of China since 1998 followed by huge economic losses. However, the full-length transcriptome information of A. sanguinea is still not fully explored, which hampers basic genetic and functional studies. Herein, single-molecule real-time (SMRT) sequencing technology was performed to characterize the full-length transcript in A. sanguinea. Totally, 83.03 Gb SMRT sequencing clean reads were generated, 983,960 circular consensus sequences (CCS) with average lengths of 3,061 bp were obtained, and 81.71% (804,016) of CCS were full-length non-chimeric reads (FLNC). Furthermore, 26,461 contigs were obtained after being corrected with Illumina library sequencing, with 20,037 (75.72%) successfully annotated in the five public databases. A total of 13,441 long non-coding RNA (lncRNA) transcripts, 3,137 alternative splicing (AS) events, 514 putative transcription factors (TFs) members from 23 TF families, and 4,397 simple sequence repeats (SSRs) were predicted, respectively. Our findings provided a sizable insights into gene sequence characteristics of A. sanguinea, which can be used as a reference sequence resource for A. sanguinea draft genome annotation, and will contribute to further molecular biology research on this harmful bloom algae.

The Long Read Transcriptome of Rice (Oryza sativa ssp. japonica var. Nipponbare) Reveals Novel Transcripts

BackgroundHigh-throughput next-generation sequencing technologies offer a powerful approach to characterizing the transcriptomes of plants. Long read sequencing has been shown to support the discovery of novel isoforms of transcripts. This approach enables the generation of full-length sequences revealing splice variants that may be important in regulating gene action. Investigation of the diversity of transcripts in the rice transcriptome including splice variants was conducted using PacBio long-read sequence data to improve the annotation of the rice genome.ResultsA cDNA library was prepared from RNA extracted from leaves, roots, seeds, inflorescences, and panicles of O. sativa ssp. japonica var Nipponbare and sequenced on a PacBio Sequel platform. This produced 346,190 non-redundant full-length non-chimeric reads (FLNC) resulting in 33,504 high-quality transcripts. Half of the transcripts were multi-exonic and entirely matched with the reference transcripts. However, 14,874 novel isoforms were also identified resulting predominantly from intron retention and at least one novel splice site. Intron retention was the prevalent alternative splicing event and exon skipping was the least observed. Of 73,659 splice junctions, 12,755 (17%) represented novel splice junctions with canonical and non-canonical intron boundaries. The complexity of the transcriptome was examined in detail for 19 starch synthesis-related genes, defining 276 spliced isoforms of which 94 splice variants were novel.ConclusionThe data reveal the great complexity of the rice transcriptome. The novel transcripts provide new insights that may be a key input in future research to improve the annotation of the rice genome.

Dynamic Changes in Ascorbic Acid Content during Fruit Development and Ripening of Actinidia latifolia (an Ascorbate-Rich Fruit Crop) and the Associated Molecular Mechanisms.

Actinidia latifolia is one of the very few kiwifruit genotypes with extremely high ascorbic acid (AsA) content. However, a transcriptome atlas of this species is lacking. The accumulation of AsA during fruit development and ripening and the associated molecular mechanisms are still poorly understood. Herein, dynamic changes in AsA content at six different stages of A. latifolia fruit development and ripening were determined. AsA content of A. latifolia fruit reached 1108.76 ± 35.26 mg 100 g−1 FW at full maturity. A high-quality, full-length (FL) transcriptome of A. latifolia was successfully constructed for the first time using third-generation sequencing technology. The transcriptome comprises 326,926 FL non-chimeric reads, 15,505 coding sequences, 2882 transcription factors, 18,797 simple sequence repeats, 3328 long noncoding RNAs, and 231 alternative splicing events. The genes involved in AsA biosynthesis and recycling pathways were identified and compared with those in different kiwifruit genotypes. The correlation between the AsA content and expression levels of key genes in AsA biosynthesis and recycling pathways was revealed. LncRNAs that participate in AsA-related gene expression regulation were also identified. Gene expression patterns in AsA biosynthesis and metabolism exhibited a trend similar to that of AsA accumulation. Overall, this study paves the way for genetic engineering to develop kiwifruits with super-high AsA content.

Combination analysis of single-molecule long-read and Illumina sequencing provides insights into the anthocyanin accumulation mechanism in an ornamental grass, Pennisetum setaceum cv. Rubrum

Combination analysis of single-molecule long-read and Illumina sequencing provide full-length transcriptome information and shed new light on the anthocyanin accumulation mechanism of Pennisetum setaceum cv. 'Rubrum'. Pennisetum setaceum cv. 'Rubrum' is an ornamental grass with purple leaves widely used in landscaping. However, the current next-generation sequencing (NGS) transcriptome information of this species is not satisfactory due to the difficulties in obtaining full-length transcripts. Furthermore, the molecular mechanisms of anthocyanin accumulation in P. setaceum have not been thoroughly studied. In this study, we used PacBio full-length transcriptome sequencing (SMRT) combined with NGS technology to build and improve the transcriptomic datasets and reveal the molecular mechanism of anthocyanin accumulation in P. setaceum cv. 'Rubrum'. Therefore, 280,413 full-length non-chimeric reads sequences were obtained using the SMRT technology. We obtained 97,450 high-quality non-redundant transcripts and identified 5352 alternative splicing events. In addition, 93,066 open reading frames (ORFs), including 57,457 full ORFs and 2910 long non-coding RNA (lncRNAs) were screened out. Furthermore, 10,795 differentially expressed genes were identified using NGS. We also explored key genes, synthesis pathways, and detected lncRNA involved in anthocyanin accumulation, providing new insights into anthocyanin accumulation in P. setaceum cv. 'Rubrum'. To our best knowledge, we provided the full-length transcriptome information of P. setaceum cv. 'Rubrum' for the first time. The results of this study will provide baseline information for gene function studies and pave the way for future P. setaceum cv. 'Rubrum' breeding projects.

Screening and Validation of p38 MAPK Involved in Ovarian Development of Brachymystax lenok

Brachymystax lenok (lenok) is a rare cold-water fish native to China that is of high meat quality. Its wild population has declined sharply in recent years, and therefore, exploring the molecular mechanisms underlying the development and reproduction of lenoks for the purposes of artificial breeding and genetic improvement is necessary. The lenok comparative transcriptome was analyzed by combining single molecule, real-time, and next generation sequencing (NGS) technology. Differentially expressed genes (DEGs) were identified in five tissues (head kidney, spleen, liver, muscle, and gonad) between immature [300 days post-hatching (dph)] and mature [three years post-hatching (ph)] lenoks. In total, 234,124 and 229,008 full-length non-chimeric reads were obtained from the immature and mature sequencing data, respectively. After NGS correction, 61,405 and 59,372 non-redundant transcripts were obtained for the expression level and pathway enrichment analyses, respectively. Compared with the mature group, 719 genes with significantly increased expression and 1,727 genes with significantly decreased expression in all five tissues were found in the immature group. Furthermore, DEGs and pathways involved in the endocrine system and gonadal development were identified, and p38 mitogen-activated protein kinases (MAPKs) were identified as potentially regulating gonadal development in lenok. Inhibiting the activity of p38 MAPKs resulted in abnormal levels of gonadotropin-releasing hormone, follicle-stimulating hormone, and estradiol, and affected follicular development. The full-length transcriptome data obtained in this study may provide a valuable reference for the study of gene function, gene expression, and evolutionary relationships in B. lenok and may illustrate the basic regulatory mechanism of ovarian development in teleosts.

Elucidating the Molecular Mechanisms by which Seed-Borne Endophytic Fungi, Epichloë gansuensis, Increases the Tolerance of Achnatherum inebrians to NaCl Stress.

Seed-borne endophyte Epichloë gansuensis enhance NaCl tolerance in Achnatherum inebrians and increase its biomass. However, the molecular mechanism by which E. gansuensis increases the tolerance of host grasses to NaCl stress is unclear. Hence, we firstly explored the full-length transcriptome information of A. inebrians by PacBio RS II. In this work, we obtained 738,588 full-length non-chimeric reads, 36,105 transcript sequences and 27,202 complete CDSs from A. inebrians. We identified 3558 transcription factors (TFs), 15,945 simple sequence repeats and 963 long non-coding RNAs of A. inebrians. The present results show that 2464 and 1817 genes were differentially expressed by E. gansuensis in the leaves of E+ and E− plants at 0 mM and 200 mM NaCl concentrations, respectively. In addition, NaCl stress significantly regulated 4919 DEGs and 502 DEGs in the leaves of E+ and E− plants, respectively. Transcripts associated with photosynthesis, plant hormone signal transduction, amino acids metabolism, flavonoid biosynthetic process and WRKY TFs were differentially expressed by E. gansuensis; importantly, E. gansuensis up-regulated biology processes (brassinosteroid biosynthesis, oxidation–reduction, cellular calcium ion homeostasis, carotene biosynthesis, positive regulation of proteasomal ubiquitin-dependent protein catabolism and proanthocyanidin biosynthesis) of host grass under NaCl stress, which indicated an increase in the ability of host grasses’ adaptation to NaCl stress. In conclusion, our study demonstrates the molecular mechanism for E. gansuensis to increase the tolerance to salt stress in the host, which provides a theoretical basis for the molecular breed to create salt-tolerant forage with endophytes.

Single molecule, full-length transcript sequencing provides insight into the TPS gene family in Paeonia ostii.

BackgroundThe tree peony (Paeonia section Moutan DC), one of the traditional famous flowers with both ornamental and medicinal value, was widely used in China. Surprisingly little is known about the full-length transcriptome sequencing in tree peony, limiting the research on its gene function and molecular mechanism. The trehalose phosphate phosphatase (TPS) family genes has been found to affect plant growth and development and the function of TPS genes in Paeonia ostii is unknown.MethodsIn our study, we performed single molecule, full-length transcript sequencing in P. ostii. 10 TPS family members were identified from PacBio sequencing for bioinformatics analysis and transcriptional expression analysis.ResultsA total of 230,736 reads of insert (ROI) sequences and 114,215 full-Length non-chimeric reads (FLNC) were obtained for further ORFs and transcription factors prediction, SSR analysis and lncRNA identification. NR, Swissprot, GO, COG, KOG, Pfam and KEGG databases were used to obtain annotation information of transcripts. 10 TPS family members were identified with molecular weights between 48.0 to 108.5 kD and isoelectric point between 5.61 to 6.37. Furthermore, we found that TPS family members contain conserved TPP or TPS domain. Based on phylogenetic tree analysis, PoTPS1 protein was highly similar to AtTPS1 protein in Arabidopsis. Finally, we analyzed the expression levels of all TPS genes in P. ostii and found PoTPS5 expressed at the highest level. In conclusion, this study combined the results of the transcriptome to systematically analyze the 10 TPS family members, and sets a framework for further research of this important gene family in development of tree peony.

Analysis of transcripts and splice isoforms in red claw crayfish (Cherax quadricarinatus) using single-molecule long-read sequencing

Red claw crayfish (Cherax quadricarinatus) has recently attracted widespread attention as an emerging candidate for sustainable aquaculture production in Australia and abroad. Although they are of great economic importance, few full-length transcriptomes are available. Here, transcripts of C. quadricarinatus were generated by using Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology. With SMRT, 289,232 full-length nonchimeric reads were identified, resulting in 14,089 unigene, with an N50 length of 4363 bp and a mean read length of 3913 bp. The error rate of SMRT sequences by comparison with Illumina-produced short reads was only 0.011%. A total of 13,449 transcripts (95.4%) were annotated against the protein database. Across all transcripts, 4952 long noncoding RNAs (lncRNAs), 1090 putative TF members and 21,108 simple sequence repeats (SSRs) were identified. After Vibrio alginolyticus infection, a total of 252 DEGs were screened out after a comparative analysis between samples. Of the 252 DEGs, Hsps, IAPs and Gpx were upregulated during V. alginolyticus infection, many genes were associated with V. cholerae infection, the NF-kappa B signalling pathway, apoptosis-multiple species, the TNF signalling pathway and antigen processing and presentation pathways. Our study provides a rich set of full-length cDNA sequences for C. quadricarinatus, which will greatly facilitate crayfish transcriptome research.

PacBio Single-Molecule Long-Read Sequencing Reveals Genes Tolerating Manganese Stress in Schima superba Saplings.

Schima superba (Theaceae) is a subtropical evergreen tree and is used widely for forest firebreaks and gardening. It is a plant that tolerates salt and typically accumulates elevated amounts of manganese in the leaves. With large ecological amplitude, this tree species grows quickly. Due to its substantial biomass, it has a great potential for soil remediation. To evaluate the thorough framework of the mRNA, we employed PacBio sequencing technology for the first time to generate S. Superba transcriptome. In this analysis, overall, 511,759 full length non-chimeric reads were acquired, and 163,834 high-quality full-length reads were obtained. Overall, 93,362 open reading frames were obtained, of which 78,255 were complete. In gene annotation analyses, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Non-Redundant (Nr) databases were allocated 91,082, 71,839, 38,914, and 38,376 transcripts, respectively. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT), and Coding Non-Coding Index (CNCI) databases and observed 8,551, 9,174, 20,720, and 18,669 lncRNAs, respectively. Moreover, nine genes were randomly selected for the expression analysis, which showed the highest expression of Gene 6 (Na_Ca_ex gene), and CAX (CAX-interacting protein 4) was higher in manganese (Mn)-treated group. This work provided significant number of full-length transcripts and refined the annotation of the reference genome, which will ease advanced genetic analyses of S. superba.

Functional Annotation of a Full-Length Transcriptome and Identification of Genes Associated with Flower Development in Rhododendronsimsii (Ericaceae).

Rhododendronsimsii is one of the top ten famous flowers in China. Due to its historical value and high aesthetic, it is widely popular among Chinese people. Various colors are important breeding objectives in Rhododendron L. The understanding of the molecular mechanism of flower color formation can provide a theoretical basis for the improvement of flower color in Rhododendron L. To generate the R. simsii transcriptome, PacBio sequencing technology has been used. A total of 833,137 full-length non-chimeric reads were obtained and 726,846 high-quality full-length transcripts were found. Moreover, 40,556 total open reading frames were obtained; of which 36,018 were complete. In gene annotation analyses, 39,411, 18,565, 16,102 and 17,450 transcriptions were allocated to GO, Nr, KEGG and COG databases, correspondingly. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with Protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT) and Coding Non Coding Index (CNCI) databases and observed 6170, 2265, 4084 and 1240 lncRNAs, respectively. Based on the results, most genes were enriched in the flavonoid biosynthetic pathway. The eight key genes on the anthocyanin biosynthetic pathway were further selected and analyzed by qRT-PCR. The F3′H and ANS showed an upward trend in the developmental stages of R. simsii. The highest expression of F3′5′H and FLS in the petal color formation of R. simsii was observed. This research provided a huge number of full-length transcripts, which will help to proceed genetic analyses of R. simsii. native, which is a semi-deciduous shrub.

Comprehensive analysis of full-length transcriptomes of Schizothorax prenanti by single-molecule long-read sequencing

Schizothorax prenanti (S. prenanti) is one of the most important aquaculture species in the southwest of China. However, information of the full-length transcripts in S. prenanti remains unknown. In this study, single-molecule real-time (SMRT) sequencing was performed to generate full-length transcriptomes of S.prenanti. In total, 23.26 Gb of clean reads were generated. A total of 312,587 circular consensus sequences (CCS) were obtained with average lengths of 2634 bp and 84.16% (270,662) of CCS were full-length non-chimeric reads. After being corrected with Illumina library sequencing, 18,005 contigs were obtained, with 17,797 (98.81%) successfully annotated in eight public databases, including 15,839 complete open reading frames (ORFs) with an average length of 1330 bp. Furthermore, a total of 4152 alternative splicing (AS) events and 250 long non-coding RNA (lncRNA) transcripts were detected. Additionally, a total of 1129 putative transcription factors (TFs) members from 56 TF families and 11,660 simple sequence repeats (SSRs) were identified. This study provided a valuable resource of full-length transcripts for further research on S. prenanti.

Full-length transcriptome analysis of Phytolacca americana and its congener P. icosandra and gene expression normalization in three Phytolaccaceae species

BackgroundPhytolaccaceae species in China are not only ornamental plants but also perennial herbs that are closely related to human health. However, both large-scale full-length cDNA sequencing and reference gene validation of Phytolaccaceae members are still lacking. Therefore, single-molecule real-time sequencing technology was employed to generate full-length transcriptome in invasive Phytolacca americana and non-invasive exotic P. icosandra. Based on the transcriptome data, RT-qPCR was employed to evaluate the gene expression stability in the two plant species and another indigenous congener P. acinosa.ResultsTotal of 19.96 Gb and 19.75 Gb clean reads of P. americana and P. icosandra were generated, including 200,857 and 208,865 full length non-chimeric (FLNC) reads, respectively. Transcript clustering analysis of FLNC reads identified 89,082 and 98,448 consensus isoforms, including 86,989 and 96,764 high-quality ones. After removing redundant reads, 46,369 and 50,220 transcripts were obtained. Based on structure analysis, total 1675 and 1908 alternative splicing variants, 25,641 and 31,800 simple sequence repeats (SSR) as well as 34,971 and 36,841 complete coding sequences were detected separately. Furthermore, 3574 and 3833 lncRNA were predicted and 41,676 and 45,050 transcripts were annotated respectively. Subsequently, seven reference genes in the two plant species and a native species P. acinosa were selected and evaluated by RT-qPCR for gene expression analysis. When tested in different tissues (leaves, stems, roots and flowers), 18S rRNA showed the highest stability in P. americana, whether infested by Spodoptera litura or not. EF2 had the most stable expression in P. icosandra, while EF1-α was the most appropriate one when attacked by S. litura. EF1-α showed the highest stability in P.acinosa, whereas GAPDH was recommended when infested by S. litura. Moreover, EF1-α was the most stable one among the three plant species whenever germinating seeds or flowers only were considered.ConclusionFull-length transcriptome of P. americana and P. icosandra were produced individually. Based on the transcriptome data, the expression stability of seven candidate reference genes under different experimental conditions was evaluated. These results would facilitate further exploration of functional and comparative genomic studies in Phytolaccaceae and provide insights into invasion success of P. americana.

PacBio single molecule long-read sequencing provides insight into the complexity and diversity of the Pinctada fucata martensii transcriptome

BackgroundThe pearl oyster Pinctada fucata martensii is an economically valuable shellfish for seawater pearl production, and production of pearls depends on its growth. To date, the molecular mechanisms of the growth of this species remain poorly understood. The transcriptome sequencing has been considered to understanding of the complexity of mechanisms of the growth of P. f. martensii. The recently released genome sequences of P. f. martensii, as well as emerging Pacific Bioscience (PacBio) single-molecular sequencing technologies, provide an opportunity to thoroughly investigate these molecular mechanisms.ResultsHerein, the full-length transcriptome was analysed by combining PacBio single-molecule long-read sequencing (PacBio sequencing) and Illumina sequencing. A total of 20.65 Gb of clean data were generated, including 574,561 circular consensus reads, among which 443,944 full-length non-chimeric (FLNC) sequences were identified. Through transcript clustering analysis of FLNC reads, 32,755 consensus isoforms were identified, including 32,095 high-quality consensus sequences. After removing redundant reads, 16,388 transcripts were obtained, and 641 fusion transcripts were derived by performing fusion transcript prediction of consensus sequences. Alternative splicing analysis of the 16,388 transcripts was performed after accounting for redundancy, and 9097 gene loci were detected, including 1607 new gene loci and 14,946 newly discovered transcripts. The original boundary of 11,235 genes on the chromosomes was corrected, 12,025 complete open reading frame sequences and 635 long non-coding RNAs (LncRNAs) were predicted, and functional annotation of 13,482 new transcripts was achieved. Two thousand three hundred eighteen alternative splicing events were detected. A total of 228 differentially expressed transcripts (DETs) were identified between the largest (L) and smallest (S) pearl oysters. Compared with the S, the L showed 99 and 129 significantly up-and down-regulated DETs, respectively. Six of these DETs were further confirmed by quantitative real-time RT-PCR (RT-qPCR) in independent experiment.ConclusionsOur results significantly improve existing gene models and genome annotations, optimise the genome structure, and in-depth understanding of the complexity and diversity of the differential growth patterns of P. f. martensii.

Single molecule real-time sequencing and RNA-seq unravel the role of long non-coding and circular RNA in the regulatory network during Nile tilapia (Oreochromis niloticus) infection with Streptococcus agalactiae

BackgroundThe tilapia aquaculture industry is facing heavy economic losses due to Streptococcus agalactiae (S. agalactiae) infections. While progress has been made in past years, the lack of a high-quality tilapia genome and transcript annotations makes systematic and comprehensive exploration for a non-coding RNA regulatory network associated with the infection process unfeasible, and it stunts further research focused on disease defense and treatment. Herein, single molecular real time sequencing (SMRT-Seq) and RNA-seq data were utilized to generate a high-quality transcript annotation. In addition, Changes in mRNA and non-coding RNA expression were also analyzed during a S. agalactiae infection in tilapia. FindingsIn total, 16.79 Gb of clean data were obtained by sequencing on six SMRT cells, with 712,294 inserts (326,645 full-length non-chimeric reads and 354,188 non-full-length reads). A total of 197,952 consensus transcripts were obtained. Additionally, 55,857 transcript sequences were acquired, with 12,297 previously annotated and 43,560 newly identified transcripts. To further examine the immune response in Oreochromis niloticus following a S. agalactiae infection, a total of 470.62 Gb of clean data was generated by sequencing a library containing 18 S. agalactiae infected tilapia samples. Of the identified genes, 9911 were newly exploited, of which 7102 were functional annotated. Furthermore, 7874 mRNAs, 1281 long non-coding RNAs (out of 21,860 long non-coding RNAs), and 61 circular RNAs (out of 1026 circular RNAs) were found to be differentially expressed during infection, with the 1026 circRNAs not previously identified in tilapia. Moreover, k-means clustering and WGCNA analyses revealed that the immune response of tilapia to a S. agalactiae infection can be divided into three stages: cytokines driven rapid immune response, energy metabolism promotion, and the production of lysosomes and phagosomes. During this response, the head kidney and spleen have synergistic effects, while maintaining independent characteristics. Finally, lncRNA-mRNA (trans and cis), lncRNA-miRNA-mRNA, and circRNA-miRNA-mRNA regulatory networks were constructed and revealed that non-coding RNA is involved in the regulation of immune-related genes. ConclusionsThis study generated a greatly-improved transcript annotation for tilapia using long-read PacBio sequencing technology, and revealed the presence of a regulatory network comprised of non-coding RNAs in Nile tilapia infected with S. agalactiae.