Full-length Transcripts Research Articles

QR-1011 restores defective ABCA4 splicing caused by multiple severe ABCA4 variants underlying Stargardt disease

Stargardt disease type 1 (STGD1), the most common form of hereditary macular dystrophy, can be caused by biallelic combinations of over 2200 variants in the ABCA4 gene. This leads to reduced or absent ABCA4 protein activity, resulting in toxic metabolite accumulation in the retina and damage of the retinal pigment epithelium and photoreceptors. Approximately 21% of all ABCA4 variants that contribute to disease influence ABCA4 pre-mRNA splicing. This emphasizes the need for therapies to restore disrupted ABCA4 splicing and halt STGD1 progression. Previously, QR-1011, an antisense oligonucleotide (AON), successfully corrected splicing abnormalities and restored normal ABCA4 protein translation in human retinal organoids carrying the prevalent disease-causing variant c.5461−10T>C in ABCA4. Here, we investigated whether QR-1011 could also correct splicing in four less common non-canonical splice site (NCSS) variants flanking ABCA4 exon 39: c.5461−8T>G, c.5461−6T>C, c.5584+5G>A and c.5584+6T>C. We administered QR-1011 and three other AONs to midigene-transfected cells and demonstrate that QR-1011 had the most pronounced effect on splicing compared to the others. Moreover, QR-1011 significantly increased full-length ABCA4 transcript levels for c.5461−8T>G and c.5584+6T>C. Splicing restoration could not be achieved in the other two variants, suggesting their more severe effect on splicing. Overall, QR-1011, initially developed for a single ABCA4 variant, exhibited potent splice correction capabilities for two additional severe NCSS variants nearby. This suggests the possibility of a broader therapeutic impact of QR-1011 extending beyond its original target and highlights the potential for treating a larger population of STGD1 patients affected by multiple severe ABCA4 variants with a single AON.

Transcriptome analysis in a humanized mouse model of familial dysautonomia reveals tissue-specific gene expression disruption in the peripheral nervous system

Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) gene. This mutation results in a tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9; Elp1Δ20/flox. This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.

Full-length circRNA sequencing method using low-input RNAs and profiling of circRNAs in MPTP-PD mice on a nanopore platform.

Considering the importance of accurate information of full-length (FL) transcripts in functional analysis, researchers prefer to develop new sequencing methods based on third-generation sequencing (TGS) rather than short-read sequencing. Several FL circRNA sequencing strategies have been developed. However, the current methods are inapplicable to low-biomass samples, since a large amount of total RNAs are acquired for circRNA enrichment before library preparation. In this work, we developed an effective method to detect FL circRNAs from a nanogram level (1-100 ng) of total RNAs based on a nanopore platform. Additionally, prior to the library preparation process, we added a series of 24 nt barcodes for each sample to reduce the cost and operating time. Using this method, we profiled circRNA expression in the striatum, hippocampus and cerebral cortex of a Parkinson's disease (PD) mouse model. Over 6% of reads were effective for FL circRNA identification in most datasets. Notably, a reduction in the RNA initial input resulted in a lower correlation between replicates and the detection efficiency for longer circRNA, but the lowest input (1 ng) was able to detect numerous FL circRNAs. Next, we systematically identified over 263 934 circRNAs in PD and healthy mice using the lower-input FL sequencing method, some of which came from 50.52% of PD-associated genes. Moreover, significant changes were observed in the circRNA expression pattern at an isoform level, and high-confidence protein translation evidence was predicted. Overall, we developed an effective method to characterize FL circRNAs from low-input samples and provide a comprehensive insight into the biological function of circRNAs in PD at an isoform level.

The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part I: Library Preparation.

Global understanding of plastid gene expression has always been impaired by its complexity. RNA splicing, editing, and intercistronic processing create multiple transcripts isoforms that can hardly be resolved using traditional molecular biology techniques. During the last decade, the wide adoption of RNA-seq-based techniques has, however, allowed an unprecedented understanding of all the different steps of chloroplast gene expression, from transcription to translation. Current strategies are nonetheless unable to identify and quantify full length transcripts isoforms, a limitation that can now be overcome using Nanopore Sequencing. We here provide a complete protocol to produce, from total leaf RNA, cDNA libraries ready for Nanopore sequencing. While most Nanopore protocols take advantage of the mRNA polyA tail we here first ligate an RNA adapter to the 3' ends of the RNAs and use it to initiate the template switching reverse transcription. The cDNA is then prepared and indexed for use with the regular Oxford Nanopore v14 chemistry. This protocol is of particular interest to researchers willing to simultaneously study the multiple post-transcriptional processes prevalent in the chloroplast.

Unraveling the molecular landscape of breast muscle development in domestic Yuzhong pigeons and European meat pigeon: Insights from Iso-seq and RNA-seq analysis.

The mechanisms governing gene regulation in domestic Yuzhong pigeon breast muscle development remain largely elusive. Here, we conducted a comparative analysis using Iso-seq and RNA-seq data from domestic Yuzhong pigeons and European meat pigeons to uncover signaling pathways and genes involved in breast muscle development. The Iso-seq data from domestic Yuzhong pigeons yielded 131,377,075 subreads, resulting in 16,587 non-redundant high-quality full-length transcripts post-correction. Furthermore, utilizing pfam, CPC, PLEK, and CPAT, we predicted 5575, 4973, 2333, and 4336 lncRNAs, respectively. Notably, several genes potentially implicated in breast muscle development were identified, including tropomyosin beta chain, myosin regulatory light chain 2, and myosin binding protein C. KEGG enrichment analysis revealed critical signaling pathways in breast muscle development, spanning carbon metabolism, biosynthesis of amino acids, glycolysis/gluconeogenesis, estrogen signaling, PI3K-AKT signaling, protein processing in the endoplasmic reticulum, oxidative phosphorylation, pentose phosphate pathway, fructose and mannose metabolism, and tight junctions. These findings offer insights into the biological processes driving breast muscle development in domestic Yuzhong pigeon, contributing to our understanding of this complex phenomenon.

Full-length transcriptome reveals the circularly polarized light response-related molecular genetic characteristics of Oratosquilla oratoria

The mantis shrimp is the only animal that can recognize circularly polarized light (CPL), but its molecular genetic characteristics are unclear. Multi-tissue level full-length (FL) transcriptome sequencing of Oratosquilla oratoria, a representative widely distributed mantis shrimp, was performed in the present study. We used comparative transcriptomics to explore the critical genes of O. oratoria selected by CPL and the GNβ gene associated with CPL signal transduction was hypothesized to be positively selected. Furthermore, the FL transcriptomes of O. oratoria compound eyes under five light conditions were sequenced and used to detect alternative splicing (AS). The ASs associated with CPL recognition mainly occurred in the LWS, ARR and TRPC regions. The number of FL transcripts with AS events and annotation information also provided evidence that O. oratoria could recognize LCPL. Additionally, 51 sequences belonging to the LWS, UV and Peropsin gene families were identified based on conserved 7tm domains. The LWS, UV and Peropsin opsins have similar 3D structures with seven domains across the cell membrane and conserved KSLRTPSN, DRY, and QAKK motifs. In conclusion, these results are undoubtedly valuable for perfecting the vision theory of O. oratoria and other mantis shrimp.

The Genome-Wide Profiling of Alternative Splicing in Willow under Salt Stress

Alternative splicing (AS) is an important post-transcriptional regulatory model that can change the normal transcript expression level and possibly result in protein diversity. In this study, we conducted the full-length transcript sequencing of Salix matsudana Koidz 9901 leaves under salt treatment using the PromethION platform. A total of 4786 AS genes (9307 AS events) were determined, accounting for 7.45% of all the transcribed genes. Of them, intron retention (IR) events accounted for the most AS events (46.05%), followed by alternative 3′ splice sites (A3SS). During salt stress, the percentage of IR events decreased, and the percentage of the others increased. Statistical results showed that 5′GG was the most common motif at the 5′ end of the intron in the AS events, and GG3′ was the most common motif at the 3′ end. 5′GG-AG3′ was the most common splice mode in the AS events. The occurrence of AS events was significantly related to the exon number, exon length, intron length, GC content, and expression abundance of the genes. During salt stress, the number of AS genes gradually increased, and they mainly participated in purine and chlorophyll metabolism, RNA transport, and autophagy. Meanwhile, the AS sites of the gene increased during salt treatment, indicating the complexity of the AS events by salt stress. A comparison of differentially expressed genes (DEGs) and differentially alternative splicing (DAS) genes during salt stress revealed that they had a different mechanism of gene expression regulation when subjected to salt stress. These results expand our knowledge of AS events and shed light on and improve our understanding of plant resistance to salt tolerance in willow.

Full-Length Transcriptome and Gene Expression Analysis of Different Ovis aries Adipose Tissues Reveals Transcript Variants Involved in Lipid Biosynthesis.

Sheep have historically been bred globally as a vital food source. To explore the transcriptome of adipose tissue and investigate key genes regulating adipose metabolism in sheep, adipose tissue samples were obtained from F1 Dorper × Hu sheep. High-throughput sequencing libraries for second- and third-generation sequencing were constructed using extracted total RNA. Functional annotation of differentially expressed genes and isoforms facilitated the identification of key regulatory genes and isoforms associated with sheep fat metabolism. SMRT-seq generated 919,259 high-accuracy cDNA sequences after filtering. Full-length sequences were corrected using RNA-seq sequences, and 699,680 high-quality full-length non-chimeric (FLNC) reads were obtained. Upon evaluating the ratio of total lengths based on FLNC sequencing, it was determined that 36,909 out of 56,316 multiple-exon isoforms met the criteria for full-length status. This indicates the identification of 330,375 full-length FLNC transcripts among the 370,114 multiple-exon FLNC transcripts. By comparing the reference genomes, 60,276 loci and 111,302 isoforms were identified. In addition, 43,423 new genes and 44,563 new isoforms were identified. The results identified 185 (3198), 394 (3592), and 83 (3286) differentially expressed genes (transcripts) between tail and subcutaneous, tail and visceral, and subcutaneous and visceral adipose tissues, respectively. Functional annotation and pathway analysis revealed the following observations. (1) Among the differentially expressed genes (DEGs) of TF and SF tissues, the downregulation of ACADL, ACSL6, and NC_056060.1.2536 was observed in SF, while FFAR4 exhibited upregulation. (2) Among the DEGs of TF and VF tissues, expressions of ACADL, ACSL6, COL1A1, COL1A2, and SCD were downregulated in VF, with upregulation of FFAR4. (3) Among SF and VF expressions of COL1A1, COL1A2, and NC_056060.1.2536 were downregulated in VF. Specific differentially expressed genes (ACADL, ACSL6, COL1A1, COL1A2, FFAR4, NC_056060.1.2536, and SCD) and transcripts (NC_056066.1.1866.16 and NC_056066.1.1866.22) were identified as relevant to fat metabolism. These results provide a dataset for further verification of the regulatory pathway associated with fat metabolism in sheep.

The H2Bub1-deposition complex is required for human and mouse cardiogenesis.

De novo variants affecting monoubiquitylation of histone H2B (H2Bub1) are enriched in human congenital heart disease. H2Bub1 is required in stem cell differentiation, cilia function, post-natal cardiomyocyte maturation and transcriptional elongation. However, how H2Bub1 affects cardiogenesis is unknown. We show that the H2Bub1-deposition complex (RNF20-RNF40-UBE2B) is required for mouse cardiogenesis and for differentiation of human iPSCs into cardiomyocytes. Mice with cardiac-specific Rnf20 deletion are embryonic lethal and have abnormal myocardium. We then analyzed H2Bub1 marks during differentiation of human iPSCs into cardiomyocytes. H2Bub1 is erased from most genes at the transition from cardiac mesoderm to cardiac progenitor cells but is preserved on a subset of long cardiac-specific genes. When H2Bub1 is reduced in iPSC-derived cardiomyocytes, long cardiac-specific genes have fewer full-length transcripts. This correlates with H2Bub1 accumulation near the center of these genes. H2Bub1 accumulation near the center of tissue-specific genes was also observed in embryonic fibroblasts and fetal osteoblasts. In summary, we show that normal H2Bub1 distribution is required for cardiogenesis and cardiomyocyte differentiation, and suggest that H2Bub1 regulates tissue-specific gene expression by increasing the amount of full-length transcripts.

DNA Polymerase Diversity Reveals Multiple Incursions of Polintons During Nematode Evolution.

Polintons are double-stranded DNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family but encode a distinct protein-primed DNA polymerase B (pPolB) that is related to homologs from a different group of Polintons present outside of the Nematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting interphylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of an HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.

Single-Molecule Real-Time Sequencing for Identifying Sexual-Dimorphism-Related Transcriptomes and Genes in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)’s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-β-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-β pathway. Female-biased genes such as Smad4, Wif1, and 17-β-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis.

Dual α-Globin and Truncated EPO Receptor Knockin Restores Hemoglobin Production in α-Thalassemia-Derived Hematopoietic Stem and Progenitor Cells

Introduction: α-thalassemia major (ATM) is an autosomal recessive disorder where all four copies of the α-globin gene are deleted. A traditionally lethal disease due to severe fetal hypoxia, this condition is now survivable due to the advent of in utero blood transfusions. However, patients still require life-long transfusions postnatally. While allogeneic hematopoietic stem cell transplant (HSCT) can provide a definitive cure, it is limited by the paucity of suitable donors, has a high morbidity and mortality due the prerequisite myeloablative regimens, and carries the risk of immune rejection and graft-versus-host disease. Here, we describe a CRISPR/AAV-mediated genome editing strategy to restore a full-length copy of the α-globin gene at the β-globin locus in ATM patient-derived HSCs to restore normal hemoglobin production to a patient's red blood cells (RBCs). Methods: We identified a Cas9 gRNA that efficiently introduced indels at the HBB locus as well as an AAV6 vector that mediated efficient integration into the start codon of HBB. After designing and testing integration cassettes, we developed two candidate vectors that insert a HBA1 transgene at the HBB locus, with one bicistronic vector that also expresses a truncated form of the erythropoietin receptor (t EPOR) to increase erythropoietic output from edited HSCs. We used this approach to edit CD34 + HSCs derived from the bone marrow of three patients with ATM. Edited cells were cultured for 16 days in RBC differentiation media. Editing rates were quantified by a custom ddPCR assay. To determine the transcriptional response to these editing strategies, bulk RNA sequencing was performed comparing wild-type cells and patient cells that underwent respective editing conditions. Lastly, α-globin protein production was quantified using tetramer and single-globin high protein liquid chromatography (HPLC). Results: While effective editing frequencies in ATM HSCs were achieved by the HBA integration vector (39% edited alleles), we observed that editing rates increased over the course of RBC differentiation in the combined HBA+t EPOR edited condition, reaching 76% edited alleles by day 16. We also found that total cell counts by the end of RBC differentiation more than doubled (2.2-fold) in the HBA+t EPOR condition compared to the HBA alone condition. RNA sequencing of edited ATM-derived HSCs following RBC differentiation revealed an upregulation of HBA1, HBA2, and EPOR transcripts compared to unedited patient cells. These results were supported by HPLC, which demonstrated restoration of hemoglobin tetramers, including fetal hemoglobin (α+γ subunits), adult hemoglobin (α+β subunits), and hemoglobin A2 (α+δ subunits) compared to unedited patient cells. Complementing this with globin single chain analysis found that we had achieved 11% restoration of α-globin compared to wild-type levels. In spite of integration at the HBB locus, neither RNA sequencing nor HPLC showed disruption of β-globin production. α-globin production with a concomitant decrease in the formation of toxic β-globin dimers was confirmed by Western blot, with restoration of the normal balance of α-globin to dimeric β-globin in edited cells to wild-type levels. Conclusions: To our knowledge, these results demonstrate for the first time that gene therapy or genome editing may be used to increase α-globin production in ATM patient-derived RBCs. We found that the editing strategy that paired site-specific α-globin integration with t EPOR expression achieved the highest editing frequencies, greatest production of edited cells, and the most robust production of α-globin protein compared to α-globin integration alone. By increasing erythropoietic output using t EPOR, this approach has the potential to overcome some of the clinical challenges previously seen in HSCT for the hemoglobinopathies, including low editing and engraftment rates. Moreover, as patients with severe anemia such those with ATM have increased levels of circulating erythropoietin due to severe anemia, a t EPOR-based enrichment strategy is likely to be especially potent following transplantation into ATM bone marrow. We believe this could allow clinicians to reduce or eliminate the need for myeloablation, which stand as a major barrier to HSCT safety and efficacy. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for ATM.

Development of 2'-O-Methyl and LNA Antisense Oligonucleotides for SMN2 Splicing Correction in SMA Cells.

Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease caused by mutations in the SMN1 gene. Existing therapies demonstrate positive results on SMA patients but still might be ameliorated in efficacy and price. In the presented study we designed antisense oligonucleotides (AONs), targeting intronic splicing silencer sites, some were modified with 2'-O-methyl, others with LNA. The AONs have been extensively tested in different concentrations, both individually and combined, in order to effectively target the ISS-N1 and A+100G splicing silencer regions in intron 7 of the SMN2 gene. By treating SMA-cultured fibroblasts with certain AONs, we discovered a remarkable increase in the levels of full-length SMN transcripts and the number of nuclear gems. This increase was observed to be dose-dependent and reached levels comparable to those found in healthy cells. When added to cells together, most of the tested molecules showed a remarkable synergistic effect in correcting splicing. Through our research, we have discovered that the impact of oligonucleotides is greatly influenced by their length, sequence, and pattern of modification.

Characterization and Expression of Cytochrome b5 Gene and Protein in Ovaries of Giant Tiger Shrimp Penaeus monodon

The full-length transcript of cytochrome b5 (PmCytb5) in the giant tiger shrimp (Penaeus monodon) was characterized. It was 1539 bp with an open reading frame (ORF) of 432 bp deducing to 143 amino acids. The deduced PmCytb5 protein contained predicted cytochrome b5-like heme/steroid binding domain (Cytb5, positions 17-91) and a transmembrane domain (positions 120-139). In non-ablated wild adults, PmCytb transcript was significantly increased in mature ovaries while it was earlier upregulated in late vitellogenic stage in eyestalk-ablated shrimp (P<0.05). Progesterone injection (0.1 μg/g) induced the expression of PmCytb5 at 48 hours post injection (hpi, P<0.05) but serotonin (50 μg/g) did not alter its expression (P>0.05) in domesticated adults. The cytoplasmic PmCytb5 protein was upregulated from immature ovaries in the early vitellogenic stage and comparably expressed in subsequent stages in non-ablated wild broodstock. In eyestalk-ablated wild broodstock, a similar expression profile was observed but its expression was further increased in late vitellogenic, and mature stages. The membrane PmCytb5 protein was upregulated in late vitellogenic and mature stages in the former but expressed at a similar level in different ovarian stages in eyestalk-ablated broodstock. Results suggested the important role of PmCytb5 in ovarian development of P. monodon.

PacBio Full-Length Transcriptome Sequencing Reveals the Mechanism of Salt Stress Response in Sonneratia apetala.

Sonneratia apetala is an essential mangrove wetland restoration tree species. Studying its molecular mechanism for salt tolerance could lay a foundation for further cultivating excellent resistant germplasm. This study used a combination of PacBio isoform sequencing (Iso-seq) and BGISEQ RNA sequencing (RNA-seq) to analyze the molecular mechanism to salt stress response of one-year-old S. apetala leaves. The growth and physiological analysis showed that physiological indexes such as growth rate, net photosynthetic rate and antioxidant enzyme activity all exhibit significant changes under salt stress. From Iso-seq, a total of 295,501 full-length transcripts, with an average length of 1418 bp, were obtained. RNA-seq produced 4712 differentially expressed genes (DEGs) as compared to a control group. Of these, 930 were identified to be co-expressed during the STEM time sequence analysis. Further, 715 and 444 co-expressed DEGs were annotated by GO and KEGG analyses, respectively. Moreover, 318 of the co-expressed DEGs were annotated as essential genes that were implicated in salt stress response of S. apetala, which were involved in transcription factors, signal transduction, hormone response, ROS homeostasis, osmotic balance, cell wall synthesis or modification. These results provide candidate targets for further characterization and offer insights into the salt-tolerant mechanism of S. apetala.

Integrating Full-Length and Second-Generation Transcriptomics to Reveal Differentially Expressed Genes Associated with the Development of Corydalis yanhusuo Tuber.

Corydalis yanhusuo is a medicinal herb in China that has been widely used to treat various kinds of pain. The tuber is the main organ of C. yanhusuo used for medicinal purposes, but changes in related genes during the development of the tuber have rarely been reported. To identify the differentially expressed genes during tuber development, C. yanhusuo full-length transcriptomic sequencing was performed using single-molecule real-time technology, and tubers at three development stages were selected for comparative transcriptome analysis. A total of 90,496 full-length non-chimeric transcripts were obtained, and 19,341 transcripts were annotated in at least one public database. A total of 9221 differentially expressed genes were identified during the swelling process of C. yanhusuo tuber. A Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis revealed that differentially expressed genes associated with a "starch and sucrose metabolism pathway", "phenylpropanoid biosynthesis pathway", "isoquinoline alkaloid biosynthesis pathway", "zeatin biosynthesis pathway", and "brassinosteroid biosynthesis pathway" were predominantly enriched. In addition, the genes involved in cell wall metabolism were potentially associated with tuber swelling. These processes regulated and were involved in C. yanhusuo tuber development. The results provide a foundation for further research on tuber formation in medicinal plants.

SINGLE-CELL TRANSCRIPTOME ANALYSIS IN HEALTH AND DISEASE.

The analysis of the single-cell transcriptome has emerged as a powerful tool to gain insights on the basic mechanisms of health and disease. It is widely used to reveal the cellular diversity and complexity of tissues at cellular resolution by RNA sequencing of the whole transcriptome from a single cell. Equally, it is applied to discover an unknown, rare population of cells in the tissue. The prime advantage of single-cell transcriptome analysis is the detection of stochastic nature of gene expression of the cell in tissue. Moreover, the availability of multiple platforms for the single-cell transcriptome has broadened its approaches to using cells of different sizes and shapes, including the capture of short or full-length transcripts, which is helpful in the analysis of challenging biological samples. And with the development of numerous packages in R and Python, new directions in the computational analysis of single-cell transcriptomes can be taken to characterize healthy versus diseased tissues to obtain novel pathological insights. Downstream analysis such as differential gene expression analysis, gene ontology term analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, cell-cell interaction analysis, and trajectory analysis has become standard practice in the workflow of single-cell transcriptome analysis to further examine the biology of different cell types. Here, we provide a broad overview of single-cell transcriptome analysis in health and disease conditions currently applied in various studies.

A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA

Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2–3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50–60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.

Comparative transcriptome analysis of dioecious floral development in Trachycarpus fortunei using Illumina and PacBio SMRT sequencing

BackgroundTrachycarpus fortunei is a plant with significant economic and ornamental value. Both male and female flowers of T. fortunei originate as bisexual flowers, and selective abortion occurs during floral development. However, the regulatory mechanisms underlying this process remain unclear in T. fortunei. In this study, transcriptome sequencing with Illumina and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) platforms were used to investigate gene expression differences between male and female T. fortunei plants.ResultsA total of 833,137 full-length non-chimeric (FLNC) reads were obtained, and 726,846 high-quality full-length transcripts were identified. A total of 159 genes were differentially expressed between male and female flowers at all development stages. Some of the differentially expressed genes (DEGs) showed male bias, including serine/threonine-protein kinase (STPK), THUMP1 homolog and other genes. Through single-nucleotide polymorphisms(SNPs) identification, 28 genes were considered as potential sex-associated SNPs. Time-Ordered Gene Co-expression Network (TO-GCN) analysis revealed that MADS2 and MADS26 may play important roles in the development of female and male flowers T. fortune plants, respectively.ConclusionsThese findings provide a genetic basis for flower development and differentiation in T. fortunei, and improve our understanding of the mechanisms underlying sexual differentiation in T. fortunei.

PacBio Full-Length Transcriptome of a Tetraploid Sinocyclocheilus multipunctatus Provides Insights into the Evolution of Cavefish.

Sinocyclocheilus multipunctatus is a second-class nationally protected wild animal in China. As one of the cavefish, S. multipunctatus has strong adaptability to harsh subterranean environments. In this study, we used PacBio SMRT sequencing technology to generate a first representative full-length transcriptome for S. multipunctatus. Sequence clustering analysis obtained 232,126 full-length transcripts. Among all transcripts, 40,487 were annotated in public databases, while 70,300 microsatellites, 2384 transcription factors, and 16,321 long non-coding RNAs were identified. The phylogenetic tree showed that S. multipunctatus shows a closer relationship to Carassius auratus and Cyprinus carpio, phylogenetically diverging from the common ancestor ~14.74 million years ago (Mya). We also found that between 15.6 and 17.5 Mya, S. multipunctatus also experienced an additional whole-genome duplication (WGD) event, which may have promoted the species evolution of S. multipunctatus. Meanwhile, the overall rates of evolutionary of polyploid S. multipunctatus were significantly higher than those of the other cyprinids, and 220 positively selected genes (PSGs) were identified in two sub-genomes of S. multipunctatus. These PSGs are likely to fulfill critical roles in the process of adapting to diverse cave environments. This study has the potential to facilitate future investigations into the genomic characteristics of S. multipunctatus and provide valuable insights into revealing the evolutionary history of polyploid S. multipunctatus.