Consensus Isoforms Research Articles

Full-Length Transcriptome Sequencing and Identification of Genes Related to Terpenoid Biosynthesis in Cinnamomum migao H. W. Li

Cinnamomum migao H. W. Li is an evergreen woody plant that is only distributed in southwest China. The volatile oils from the fruits of C. migao have long been used as a special authentic medicinal herb by local ethnic minorities. Due to its low seed germination rate, destructive lumbering and low rates of artificial planting, C. migao is facing the danger of extinction. Therefore, it is urgent to exploit and protect this species using molecular biological technology, especially to target the genes involved in the biosynthesis of terpenoids in the volatile oil. However, the genomic data for this plant are not available. In this study, the transcriptome sequencing of C. migao was performed to obtain the key genes involved in terpenoid biosynthesis via a combination of full-length transcriptome and next-generation sequencing (NGS). More than 39.9 Gb of raw data was obtained and 515,929 circular consensus sequences (CCSs) were extracted. After clustering similar 472,858 full-length non-chimeric (FLNC) reads and correction with NGS data, 139,871 consensus isoforms were obtained. Meanwhile, 73,575 non-redundant transcripts were generated by removing redundant transcripts. Then, 70,427 isoforms were successfully annotated using public databases. Moreover, differentially expressed transcripts (DETs) in four different developmental stages of the C. migao fruit were analyzed and 5764 transcripts showed period-specific expression. Finally, 15 transcripts, 6 transcripts, and only 1 transcript were identified as being involved in the biosynthesis of sesquiterpenoids, diterpenoids, and monoterpenoids, respectively. This study provides a basis for future research in the gene mining, genetic breeding, and metabolic engineering of C. migao.

De novo transcriptome assembly of Iphiculus spongiosus Adams & White, 1849 (Decapoda; Brachyura; Leucosioidea) using full-length isoform sequencing

Brachyura play a key role in linking food energy transfer in marine benthic ecosystems. Hence, the health and sustainability of benthic ecosystems come from the maintenance of crab diversity, and systematic studies should be performed. Recently, although mitochondrial genomes have been widely used in brachyuran phylogeny, the systematic reconstruction of leucosiid crabs has mainly depended on museum specimens due to the lack of live specimens and reference databases. Here, therefore, the selected ribonucleic acid (RNA) was isolated from Iphiculus spongiosus for long-read, single molecule sequencing based on the PacBio full-length isoform sequencing (ISO-seq) platform. A total of 173,863 high-quality consensus isoforms were obtained with a total sequencing length of 210,170,210 bp. Performing functional annotation by including seven general databases revealed a total of 73,809 transcripts (based on the identified coding region sequences), of which the total length was 56,483,844 bp. These results represent the first RNA sequencing data of the leucosiid group. They can contribute to the de novo transcriptome assembly of a reference database and could aid the development of genetic probes to aid target sequence capture in future taxonomic studies.

Full-Length Transcriptome Analysis of the Halophyte Nitraria sibirica Pall.

Background: Nitraria sibirica Pall. is one of the pioneer tree species in saline–alkali areas due to its extreme salt tolerance. However, the lack of information on its genome limits the further exploration of the molecular mechanisms in N. sibirica under salt stress. Methods: In this study, we used single-molecule real-time (SMRT) technology based on the PacBio Iso-Seq platform to obtain transcriptome data from N. sibirica under salt treatment for the first time, which is helpful for our in-depth analysis of the salt tolerance and molecular characteristics of N. sibirica. Results: Our results suggested that a total of 234,508 circular consensus sequences (CCSs) with a mean read length of 2121 bp were obtained from the 19.26 Gb raw data. Furthermore, based on transcript cluster analysis, 93,713 consensus isoforms were obtained, including 92,116 high-quality isoforms. After removing redundant sequences, 49,240 non-redundant transcripts were obtained from high-quality isoforms. A total of 37,261 SSRs, 1816 LncRNAs and 47,314 CDSs, of which 40,160 carried complete ORFs, were obtained. Based on our transcriptome data, we also analyzed the coding genes of H+-PPase, and the results of both bioinformatics and functional analyses indicated that the gene prediction via full-length transcripts obtained by SMRT technology is reliable and effective. In summary, our research data obtained by SMRT technology provides more reliable and accurate information for the further analysis of the regulatory network and molecular mechanism of N. sibirica under salt stress.

Improved Reference Genome Annotation of Brassica rapa by Pacific Biosciences RNA Sequencing.

The species Brassica rapa includes several important vegetable crops. The draft reference genome of B. rapa ssp. pekinensis was completed in 2011, and it has since been updated twice. The pangenome with structural variations of 18 B. rapa accessions was published in 2021. Although extensive genomic analysis has been conducted on B. rapa, a comprehensive genome annotation including gene structure, alternative splicing (AS) events, and non-coding genes is still lacking. Therefore, we used the Pacific Biosciences (PacBio) single-molecular long-read technology to improve gene models and produced the annotated genome version 3.5. In total, we obtained 753,041 full-length non-chimeric (FLNC) reads and collapsed these into 92,810 non-redundant consensus isoforms, capturing 48% of the genes annotated in the B. rapa reference genome annotation v3.1. Based on the isoform data, we identified 830 novel protein-coding genes that were missed in previous genome annotations, defined the untranslated regions (UTRs) of 20,340 annotated genes and corrected 886 wrongly spliced genes. We also identified 28,564 AS events and 1,480 long non-coding RNAs (lncRNAs). We produced a relatively complete and high-quality reference transcriptome for B. rapa that can facilitate further functional genomic research.

Comparative Transcriptomic Analyses Provide Insights into the Enzymatic Browning Mechanism of Fresh-Cut Sand Pear Fruit

Pear (Pyrus spp.) is one of the most commonly consumed temperate fruits, having considerable economic and health importance. Fresh-cut or processed pear fruits are prone to browning because of the abundant phenolic compounds; however, little is known about the molecular mechanisms underlying enzymatic browning of fresh-cut sand pear fruit. In this study, fruits of two sand pear genotypes (low browning cultivar ‘Eli No.2′ and high browning cultivar ‘Weiningdahuangli’) were used to analyze the molecular mechanism of enzymatic browning by SMRT-seq and RNA-seq. The results generated 69,122 consensus isoforms, 21,336 new transcripts, 7105 alternative splicing events, and 254 long non-coding RNAs (lncRNAs). Furthermore, five genes related to enzymatic browning were predicted to be targets of six lncRNAs, and 9930 differentially expressed genes (DEGs) were identified between two different flesh browning cultivars. Meanwhile, most DEGs (e.g., PAL, 4CL, CAD, CCR, CHS, and LAR) involved in the phenylpropanoid biosynthesis pathway were up-regulated, and the expression of PPO and POD were highly expressed in the high-browning cultivar. Interestingly, the transcript level of PbrPPO4 (Pbr000321.4) was significantly higher than other PPO and POD genes, and a high level of total polyphenol and PPO activity were observed in the high browning cultivar. We found that the expression of lncRNA PB.156.1 was significantly positively correlated with the target gene PbrPPO4 (Pbr000321.4). The results suggest that PbrPPO4 might act as a major contributor and a key enzyme encoding gene in regulating fresh-cut sand pear fruit enzymatic browning; the expression of PbrPPO4 was probably regulated by lncRNA PB.156.1. Altogether, the transcriptomic and physiological analyses expand the knowledge of sand pear flesh enzymatic browning at the molecular level and provide a foundation for germplasm resources for molecular breeding of high polyphenol and low browning cultivars in sand pears.

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

BackgroundEimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix.MethodsSecond-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied.ResultsSMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2.ConclusionsWe obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control.Graphical

The Landscapes of Full-Length Transcripts and Splice Isoforms as Well as Transposons Exonization in the Lepidopteran Model System, Bombyx mori.

The domesticated silkworm, Bombyx mori, is an important model system for the order Lepidoptera. Currently, based on third-generation sequencing, the chromosome-level genome of Bombyx mori has been released. However, its transcripts were mainly assembled by using short reads of second-generation sequencing and expressed sequence tags which cannot explain the transcript profile accurately. Here, we used PacBio Iso-Seq technology to investigate the transcripts from 45 developmental stages of Bombyx mori. We obtained 25,970 non-redundant high-quality consensus isoforms capturing ∼60% of previous reported RNAs, 15,431 (∼47%) novel transcripts, and identified 7,253 long non-coding RNA (lncRNA) with a large proportion of novel lncRNA (∼56%). In addition, we found that transposable elements (TEs) exonization account for 11,671 (∼45%) transcripts including 5,980 protein-coding transcripts (∼32%) and 5,691 lncRNAs (∼79%). Overall, our results expand the silkworm transcripts and have general implications to understand the interaction between TEs and their host genes. These transcripts resource will promote functional studies of genes and lncRNAs as well as TEs in the silkworm.

Full-Length Transcriptome Analysis Reveals Candidate Genes Involved in Terpenoid Biosynthesis in Artemisia argyi.

Artemisia argyi is an important medicinal plant widely utilized for moxibustion heat therapy in China. The terpenoid biosynthesis process in A. argyi is speculated to play a key role in conferring its medicinal value. However, the molecular mechanism underlying terpenoid biosynthesis remains unclear, in part because the reference genome of A. argyi is unavailable. Moreover, the full-length transcriptome of A. argyi has not yet been sequenced. Therefore, in this study, de novo transcriptome sequencing of A. argyi's root, stem, and leaf tissues was performed to obtain those candidate genes related to terpenoid biosynthesis, by combining the PacBio single-molecule real-time (SMRT) and Illumina sequencing NGS platforms. And more than 55.4 Gb of sequencing data and 108,846 full-length reads (non-chimeric) were generated by the Illumina and PacBio platform, respectively. Then, 53,043 consensus isoforms were clustered and used to represent 36,820 non-redundant transcripts, of which 34,839 (94.62%) were annotated in public databases. In the comparison sets of leaves vs roots, and leaves vs stems, 13,850 (7,566 up-regulated, 6,284 down-regulated) and 9,502 (5,284 up-regulated, 4,218 down-regulated) differentially expressed transcripts (DETs) were obtained, respectively. Specifically, the expression profile and KEGG functional enrichment analysis of these DETs indicated that they were significantly enriched in the biosynthesis of amino acids, carotenoids, diterpenoids and flavonoids, as well as the metabolism processes of glycine, serine and threonine. Moreover, multiple genes encoding significant enzymes or transcription factors related to diterpenoid biosynthesis were highly expressed in the A. argyi leaves. Additionally, several transcription factor families, such as RLK-Pelle_LRR-L-1 and RLK-Pelle_DLSV, were also identified. In conclusion, this study offers a valuable resource for transcriptome information, and provides a functional genomic foundation for further research on molecular mechanisms underlying the medicinal use of A. argyi leaves.

Full-length transcriptome sequence analysis of Eimeria necatrix unsporulated oocysts and sporozoites identifies genes involved in cellular invasion

Eimeria necatrix is one of the most pathogenic chicken coccidia and causes avian coccidiosis, an enteric disease of major economic importance worldwide. Eimeria parasites have complex developmental life cycles, with an exogenous phase in the environment and an endogenous phase in the chicken intestine. Oocysts excreted by chickens rapidly undergo meiosis and cell division to form eight haploid sporozoites (SZ). SZ liberated from sporocysts in the chicken intestine migrate to their preferred site of development to initiate cellular invasion. To date, almost nothing is known about the proteins that mediate parasite invasion in E. necatrix. In order to discover genes with functions involved in cellular invasion, the transcriptome profiles of E. necatrix unsporulated oocysts (UO) and SZ were analyzed using a combination of third-generation single-molecule real-time sequencing (TGS) and second-generation sequencing (SGS) followed by qRT-PCR validation. Correction of TGS long reads by SGS short reads resulted in 34,932 (UO) and 23,040 (SZ) consensus isoforms. After subsequent assembly, a total of 4949 and 4254 genes were identified from UO and SZ libraries, respectively. A total of 8376 genes were identified as differentially expressed genes (DEGs) between SZ and UO. Compared to UO, 4057 genes were upregulated and 4319 genes were downregulated in SZ. Approximately 1399 and 1758 genes were defined as stage-specific genes in SZ and UO, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 2978 upregulated SZ genes were clustered into 29 GO terms, and 857 upregulated SZ genes were associated with 26 KEGG pathways. We also predicted a further 50 upregulated SZ genes and 73 upregulated UO genes encoding microneme proteins, apical membrane antigens, rhoptry neck proteins, rhoptry proteins, dense granule proteins, heat shock proteins, calcium-dependent protein kinases, cyclin-dependent kinases, cGMP-dependent protein kinase, and glycosylphosphatidylinositol-anchored surface antigens. Our data reveal new features of the E. necatrix transcriptional landscape and provide resources for the development of novel vaccine candidates against E. necatrix infection.

Transcriptomic and Proteomic Analyses of Nepenthes ampullaria and Nepenthes rafflesiana Reveal Parental Molecular Expression in the Pitchers of Their Hybrid, Nepenthes × hookeriana.

Nepenthes is a genus comprising carnivorous tropical pitcher plants that have evolved trapping organs at the tip of their leaves for nutrient acquisition from insect trapping. Recent studies have applied proteomics approaches to identify proteins in the pitcher fluids for better understanding the carnivory mechanism, but protein identification is hindered by limited species-specific transcriptomes for Nepenthes. In this study, the proteomics informed by transcriptomics (PIT) approach was utilized to identify and compare proteins in the pitcher fluids of Nepenthes ampullaria, Nepenthes rafflesiana, and their hybrid Nepenthes × hookeriana through PacBio isoform sequencing (Iso-Seq) and liquid chromatography-mass spectrometry (LC-MS) proteomic profiling. We generated full-length transcriptomes from all three species of 80,791 consensus isoforms with an average length of 1,692 bp as a reference for protein identification. The comparative analysis found that transcripts and proteins identified in the hybrid N. × hookeriana were more resembling N. rafflesiana, both of which are insectivorous compared with omnivorous N. ampullaria that can derive nutrients from leaf litters. Previously reported hydrolytic proteins were detected, including proteases, glucanases, chitinases, phosphatases, nucleases, peroxidases, lipid transfer protein, thaumatin-like protein, pathogenesis-related protein, and disease resistance proteins. Many new proteins with diverse predicted functions were also identified, such as amylase, invertase, catalase, kinases, ligases, synthases, esterases, transferases, transporters, and transcription factors. Despite the discovery of a few unique enzymes in N. ampullaria, we found no strong evidence of adaptive evolution to produce endogenous enzymes for the breakdown of leaf litter. A more complete picture of digestive fluid protein composition in this study provides important insights on the molecular physiology of pitchers and carnivory mechanism of Nepenthes species with distinct dietary habits.

Single-Molecule Real-Time Transcript Sequencing of Turnips Unveiling the Complexity of the Turnip Transcriptome.

To generate the full-length transcriptome of Xinjiang green and purple turnips, Brassica rapa var. Rapa, using single-molecule real-time (SMRT) sequencing. The samples of two varieties of Brassica rapa var. Rapa at five developmental stages were collected and combined to perform SMRT sequencing. Meanwhile, next generation sequencing was performed to correct SMRT sequencing data. A series of analyses were performed to investigate the transcript structure. Finally, the obtained transcripts were mapped to the genome of Brassica rapa ssp. pekinesis Chiifu to identify potential novel transcripts. For green turnip (F01), a total of 19.54 Gb clean data were obtained from 8 cells. The number of reads of insert (ROI) and full-length non-chimeric (FLNC) reads were 510,137 and 267,666. In addition, 82,640 consensus isoforms were obtained in the isoform sequences clustering, of which 69,480 were high-quality, and 13,160 low-quality sequences were corrected using Illumina RNA seq data. For purple turnip (F02), there were 20.41 Gb clean data, 552,829 ROIs, and 274,915 FLNC sequences. A total of 93,775 consensus isoforms were obtained, of which 78,798 were high-quality, and the 14,977 low-quality sequences were corrected. Following the removal of redundant sequences, there were 46,516 and 49,429 non-redundant transcripts for F01 and F02, respectively; 7,774 and 9,385 alternative splicing events were predicted for F01 and F02; 63,890 simple sequence repeats, 59,460 complete coding sequences, and 535 long-non coding RNAs were predicted. Moreover, 5,194 and 5,369 novel transcripts were identified by mapping to Brassica rapa ssp. pekinesis Chiifu. The obtained transcriptome data may improve turnip genome annotation and facilitate further study of the Brassica rapa var. Rapa genome and transcriptome.

Full-length transcriptome sequencing combined with RNA-seq analysis revealed the immune response of fat greenling (Hexagrammos otakii) to Vibrio harveyi in early infection

Fat greenling (Hexagrammos otakii) is an important commercial marine fish species cultured in northeast Asia, but its available gene sequences are limited. Vibrio harveyi is a causative agent of vibriosis in fat greenling and also causes severe losses to the aquaculture industry in China. In order to obtain more high-quality transcript information and investigate the early immune response of fat greenling against V. harveyi, the fish were artificially infected with V. harveyi, and five sampling points were set within 48 h. Iso-Seq combined with RNA-Seq were applied in the comprehensive transcriptome analysis of V. harveyi-infected fat greenling. Total 42,225 consensus isoforms were successfully extracted from the result of Iso-Seq, and more than 19,000 ORFs were predicted. In addition, total three modules were identified by WGCNA which significantly positive correlated to the infection time, and the KEGG analysis showed that the immune-related genes in these modules mainly enriched in TLR signaling pathway, NF-κB signaling pathway and Endocytosis. The activation of inflammation and endocytosis was the most significant characteristics of fat greenling immune response during the early infection. Based on the WGCNA, a series of high–degree nodes in the networks were identified as hub genes. The protein structures of cold-inducible RNA-binding protein (CIRBP), poly [ADP-ribose] polymerase 1 (PARP1) and protein arginine N-methyl transferase 1 (PRMT1) were subsequently found to be highly conserved in vertebrate, and the gene expression pattern of CIRBP, PARP1, PRMT1 and a part of TLR/NF-κB pathway-related genes indicated that these proteins might have similar biological functions in regulation of inflammatory response in teleost fish. The results of this study provided the first systematical full-length transcriptome profile of fat greenling and characterized its immune responses in early infection of V. harvey, which will serve as the foundation for further exploring the molecular mechanism of immune defense against bacterial infection in fat greenling.

Single-molecule real-time sequencing of the full-length transcriptome of loquat under low-temperature stress.

In this study, third-generation full-length (FL) transcriptome sequencing was performed of loquat using single-molecule real-time(SMRT) sequencing from the pooled cDNA of embryos of young loquat fruit under different low temperatures (three biological replicates for treatments of 1°C, -1°C, and -3°C, for 12 h or 24 h) and the control group(three biological replicates for treatments of room temperature), Illumina sequencing was used to correct FL transcriptome sequences. A total of 3 PacBio Iso-Seq libraries (1–2 kb, 2–3 kb and 3–6 kb) and 21 Illumina transcriptome libraries were constructed, a total of 13.41 Gb of clean reads were generated, which included 215,636 reads of insert (ROIs) and 121,654 FL, non-chimaric (FLNC) reads. Transcript clustering analysis of the FLNC reads revealed 76,586 consensus isoforms, and a total of 12,520 high-quality transcript sequences corrected with non-FL sequences were used for subsequent analysis. After the redundant reads were removed, 38,435 transcripts were obtained. A total of 27,905 coding DNA sequences (CDSs) were identified, and 407 long non-coding RNAs (lncRNAs) were ultimately predicted. Additionally, 24,832 simple sequence repeats (SSRs) were identified, and a total of 1,295 alternative splicing (AS) events were predicted. Furthermore, 37,993 transcripts were annotated in eight functional databases. This is the first study to perform SMRT sequencing of the FL transcriptome of loquat. The obtained transcriptomic data are conducive for further exploration of the mechanism of loquat freezing injury and thus serve as an important theoretical basis for generating new loquat material and for identifying new ways to improve loquat cold resistance.

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.

Full-length transcriptome analysis of Misgurnus anguillicaudatus.

Faithful analysis of transcripts represents a critical component of the gene discovery, genomic characterization and annotation process in species without a reference genome. The present study generated 28,001 full-length transcripts of Misgurnus anguillicaudatus using the Pacific Bioscience (PacBio) single-molecule real-time (SMRT) sequencing system. A total of 77,346 consensus isoforms were identified, and 18,991 complete open reading frames (ORFs) detected. In total, 1345 lncRNAs were identified with high-confidence, with additional identification of a number of well-known transcription factors. The present study may facilitate additional exploration of the genomic signatures of M. anguillicaudatus.

Transcriptome Profiling Provides Insight into the Genes in Carotenoid Biosynthesis during the Mesocarp and Seed Developmental Stages of Avocado (Persea americana).

Avocado (Persea americana Mill.) is an economically important crop because of its high nutritional value. However, the absence of a sequenced avocado reference genome has hindered investigations of secondary metabolism. For next-generation high-throughput transcriptome sequencing, we obtained 365,615,152 and 348,623,402 clean reads as well as 109.13 and 104.10 Gb of sequencing data for avocado mesocarp and seed, respectively, during five developmental stages. High-quality reads were assembled into 100,837 unigenes with an average length of 847.40 bp (N50 = 1725 bp). Additionally, 16,903 differentially expressed genes (DEGs) were detected, 17 of which were related to carotenoid biosynthesis. The expression levels of most of these 17 DEGs were higher in the mesocarp than in the seed during five developmental stages. In this study, the avocado mesocarp and seed transcriptome were also sequenced using single-molecule long-read sequencing to acquired 25.79 and 17.67 Gb clean data, respectively. We identified 233,014 and 238,219 consensus isoforms in avocado mesocarp and seed, respectively. Furthermore, 104 and 59 isoforms were found to correspond to the putative 11 carotenoid biosynthetic-related genes in the avocado mesocarp and seed, respectively. The isoform numbers of 10 out of the putative 11 genes involved in the carotenoid biosynthetic pathway were higher in the mesocarp than those in the seed. Besides, alpha- and beta-carotene contents in the avocado mesocarp and seed during five developmental stages were also measured, and they were higher in the mesocarp than in the seed, which validated the results of transcriptome profiling. Gene expression changes and the associated variations in gene dosage could influence carotenoid biosynthesis. These results will help to further elucidate carotenoid biosynthesis in avocado.

Full-Length Transcriptome Survey and Expression Analysis of Cassia obtusifolia to Discover Putative Genes Related to Aurantio-Obtusin Biosynthesis, Seed Formation and Development, and Stress Response.

The seed is the pharmaceutical and breeding organ of Cassia obtusifolia, a well-known medical herb containing aurantio-obtusin (a kind of anthraquinone), food, and landscape. In order to understand the molecular mechanism of the biosynthesis of aurantio-obtusin, seed formation and development, and stress response of C. obtusifolia, it is necessary to understand the genomics information. Although previous seed transcriptome of C. obtusifolia has been carried out by short-read next-generation sequencing (NGS) technology, the vast majority of the resulting unigenes did not represent full-length cDNA sequences and supply enough gene expression profile information of the various organs or tissues. In this study, fifteen cDNA libraries, which were constructed from the seed, root, stem, leaf, and flower (three repetitions with each organ) of C. obtusifolia, were sequenced using hybrid approach combining single-molecule real-time (SMRT) and NGS platform. More than 4,315,774 long reads with 9.66 Gb sequencing data and 361,427,021 short reads with 108.13 Gb sequencing data were generated by SMRT and NGS platform, respectively. 67,222 consensus isoforms were clustered from the reads and 81.73% (61,016) of which were longer than 1000 bp. Furthermore, the 67,222 consensus isoforms represented 58,106 nonredundant transcripts, 98.25% (57,092) of which were annotated and 25,573 of which were assigned to specific metabolic pathways by KEGG. CoDXS and CoDXR genes were directly used for functional characterization to validate the accuracy of sequences obtained from transcriptome. A total of 658 seed-specific transcripts indicated their special roles in physiological processes in seed. Analysis of transcripts which were involved in the early stage of anthraquinone biosynthesis suggested that the aurantio-obtusin in C. obtusifolia was mainly generated from isochorismate and Mevalonate/methylerythritol phosphate (MVA/MEP) pathway, and three reactions catalyzed by Menaquinone-specific isochorismate synthase (ICS), 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate (IPPS) might be the limited steps. Several seed-specific CYPs, SAM-dependent methyltransferase, and UDP-glycosyltransferase (UDPG) supplied promising candidate genes in the late stage of anthraquinone biosynthesis. In addition, four seed-specific transcriptional factors including three MYB Transcription Factor (MYB) and one MADS-box Transcription Factor (MADS) transcriptional factors) and alternative splicing might be involved with seed formation and development. Meanwhile, most members of Hsp20 genes showed high expression level in seed and flower; seven of which might have chaperon activities under various abiotic stresses. Finally, the expressional patterns of genes with particular interests showed similar trends in both transcriptome assay and qRT-PCR. In conclusion, this is the first full-length transcriptome sequencing reported in Caesalpiniaceae family, and thus providing a more complete insight into aurantio-obtusin biosynthesis, seed formation and development, and stress response as well in C. obtusifolia.

SMRT sequencing of full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt)

This study was aimed at generating the full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt) using single-molecule real-time (SMRT) sequencing. Four developmental stages of A. hygrophila, including eggs, larvae, pupae, and adults were harvested for isolating total RNA. The mixed samples were used for SMRT sequencing to generate the full-length transcriptome. Based on the obtained transcriptome data, alternative splicing event, simple sequence repeat (SSR) analysis, coding sequence prediction, transcript functional annotation, and lncRNA prediction were performed. Total 9.45 Gb of clean reads were generated, including 335,045 reads of insert (ROI) and 158,085 full-length non-chimeric (FLNC) reads. Transcript clustering analysis of FLNC reads identified 40,004 consensus isoforms, including 31,015 high-quality ones. After removing redundant reads, 28,982 transcripts were obtained. Total 145 alternative splicing events were predicted. Additionally, 12,753 SSRs and 16,205 coding sequences were identified based on SSR analysis. Furthermore, 24,031 transcripts were annotated in eight functional databases, and 4,198 lncRNAs were predicted. This is the first study to perform SMRT sequencing of the full-length transcriptome of A. hygrophila. The obtained transcriptome may facilitate further exploration of the genetic data of A. hygrophila and uncover the interactions between this insect and the ecosystem.

Microsatellites from Fosterella christophii (Bromeliaceae) by de novo transcriptome sequencing on the Pacific Biosciences RS platform.

Premise of the study:Microsatellite markers were developed in Fosterella christophii (Bromeliaceae) to investigate the genetic diversity and population structure within the F. micrantha group, comprising F. christophii, F. micrantha, and F. villosula.Methods and Results:Full-length cDNAs were isolated from F. christophii and sequenced on a Pacific Biosciences RS platform. A total of 1590 high-quality consensus isoforms were assembled into 971 unigenes containing 421 perfect microsatellites. Thirty primer sets were designed, of which 13 revealed a high level of polymorphism in three populations of F. christophii, with four to nine alleles per locus. Each of these 13 loci cross-amplified in the closely related species F. micrantha and F. villosula, with one to six and one to 11 alleles per locus, respectively.Conclusions:The new markers are promising tools to study the population genetics of F. christophii and to discover species boundaries within the F. micrantha group.