A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.

Characterizing the Major Structural Variant Alleles of the Human Genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.

The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages.

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases.

HDAC3 ensures stepwise epidermal stratification via NCoR/SMRT-reliant mechanisms independent of its histone deacetylase activity.

Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.

Genomics in the long-read sequencing era.

Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.

Chromosome-scale de novo genome assembly and annotation of three representative Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana.

Australian pine (Casuarina spp.) is extensively planted in tropical and subtropical regions for wood production, shelterbelts, environmental protection, and ecological restoration due to their superior biological characteristics, such as rapid growth, wind and salt tolerance, and nitrogen fixation. To analyze the genomic diversity of Casuarina, we sequenced the genomes and constructed de novo genome assemblies of the three most widely planted Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana. We generated chromosome-scale genome sequences using both Pacific Biosciences (PacBio) Sequel sequencing and chromosome conformation capture technology (Hi-C). The total genome sizes for C. equisetifolia, C. glauca, and C. cunninghamiana are 268 942 579 bp, 296 631 783 bp, and 293 483 606 bp, respectively, of which 25.91, 27.15, and 27.74% were annotated as repetitive sequences. We annotated 23 162, 24 673, and 24 674 protein-coding genes in C. equisetifolia, C. glauca, and C. cunninghamiana, respectively. We then collected branchlets from male and female individuals for whole-genome bisulfite sequencing (BS-seq) to explore the epigenetic regulation of sex determination in these three species. Transcriptome sequencing (RNA-seq) revealed differential expression of phytohormone-related genes between male and female plants. In summary, we generated three chromosome-level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.

HPV genotyping in clinical samples using long-read single-molecule real-time sequencing.

Human papillomavirus (HPV) genotyping is widely used, particularly in combination with high-risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real-time sequencing (SMRT) and evaluated its performance against MY09-11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09-11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR-HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR-HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

Molecular mechanism of Serratia marcescens Bizio infection in Reticulitermes chinensis Snyder based on full-length SMRT transcriptome sequencing.

Reticulitermes chinensis Snyder is an important pest in forestry and construction and is widely distributed in China. We found that Serratia marcescens Bizio strain SM1 has insecticidal activity to R. chinensis, but the pathogenic mechanism of SM1 to R. chinensis is not clear. Therefore, full-length transcriptome sequencing was performed on R. chinensis infected with SM1 and the control group. A total of 230 differentially expressed genes were identified by comparing SM1 infection group and the control group, among which 103 were downregulated and 127 were upregulated. We found downregulated genes in nine metabolic pathway categories, among which carbohydrate metabolism had the most downregulated genes, followed by energy metabolism and amino acid metabolism. We also found that some downregulated genes were related to pattern recognition receptors, cellular immunity, and humoral immunity, indicating that R. chinensis immunity was negatively affected by SM1 infection. In addition, some genes in signal transduction and genetic information processing pathways were downregulated. In this study, high-throughput full-length transcriptome analysis was used to analyse the pathogenic mechanism of SM1 to R. chinensis. The results of this study provide useful information for exploring the relationship between SM1 and R. chinensis, and provide theoretical support for the future application of SM1 and the prevention and treatment of R. chinensis.

Species-level understanding of the bacterial community in Daqu based on full-length 16S rRNA gene sequences.

Daqu is used as the fermentation starter of Baijiu and contributes diversified functional microbes for saccharifying grains and converting sugars into ethanol and aroma components in Baijiu products. Daqu is mainly classified into three types, namely low (LTD), medium (MTD) and high (HTD) temperature Daqu, according to the highest temperatures reached in their fermentation processes. In this study, we used the PacBio small-molecule real-time (SMRT) sequencing technology to determine the full-length 16 S rRNA gene sequences from the metagenomes of 296 samples of different types of Daqu collected from ten provinces in China, and revealed the bacterial diversity at the species level in the Daqu samples. We totally identified 310 bacteria species, including 78 highly abundant species (with a relative abundance >0.1% each) which accounted for 91.90% of the reads from all the Daqu samples. We also recognized the differentially enriched bacterial species in different types of Daqu, and in the Daqu samples with the same type but from different provinces. Specifically, Lactobacillales, Enterobacterales and Bacillaceae were significantly enriched in the LTD, MTD and HTD groups, respectively. The potential co-existence and exclusion relationships among the bacteria species involved in all the Daqu samples and in the LTD, MTD and HTD samples from a specific region were also identified. These results provide a better understanding of the bacterial diversity in different types of Daqu at the species level.

First Report of Filipino ß0-Thalassemia/ß-Thalassemia in a Chinese Family.

A pregnant woman living in Fujian Province, southeastern China, presented due to a risk of having a baby with ß-thalassemia major, during her second pregnancy, since she and her husband were suspected as ß-thalassemia carriers and their affected daughter was a transfusion-dependent patient. Using the common α-thalassemia and ß-thalassemia genotypes test, the pregnant woman was diagnosed as a ß-thalassemia carrier with ßIVS-2 - 654 (C→T)/ßN genotype and her daughter had a homozygosity for IVS - 2 - 654 (C→T) mutation, however, no abnormalities were detected in her husband. SMRT identified a Filipino ß0-deletion in her husband, and MLPA also revealed an unknown deletion in the HBB gene. Electrophoresis showed approximately 350 bp of the PCR product, and the ß-Filipino genotype presented novel fracture fragments ranging from 5,112,884 to 5,231,358 bp, and lacked a 118,475 bp fragment relative to the wild-type sequence. The daughter was therefore diagnosed with the ßIVS-2 - 654 (C→T)/ßFilipino genotype. Prenatal diagnosis with umbilical cord blood at 27th week of gestation showed heteroztgosity for IVS - 2 - 654 (C→T) mutation in the fetus and continued pregnancy was recommended. In conclusion, we identified the Filipino ß0-deletion in a Chinese family, from Fujian area, for the first time, during prenatal screening.

Integrated Untargeted Metabolome, Full-Length Sequencing and Transcriptome Analyses Reveal the Mechanism of Flavonoid Biosynthesis in Blueberry (Vaccinium spp.) Fruit.

As a highly economic berry fruit crop, blueberry is enjoyed by most people and has various potential health benefits, many of which are attributed to the relatively high concentrations of flavonoids. To obtain more accurate and comprehensive transcripts, the full-length transcriptome of half-highbush blueberry (Vaccinium corymbosum/angustifolium cultivar Northland) obtained using single molecule real-time and next-generation sequencing technologies was reported for the first time. Overall, 147,569 consensus transcripts (average length, 2738 bp; N50, 3176 bp) were obtained. After quality control steps, 63,425 high-quality isoforms were obtained and 5030 novel genes, 3002 long non-coding RNAs, 3946 transcription factor genes (TFs), 30,540 alternative splicing events, and 2285 fusion gene pairs were identified. To better explore the molecular mechanism of flavonoid biosynthesis in mature blueberry fruit, an integrative analysis of the metabolome and transcriptome was performed on the exocarp, sarcocarp, and seed. A relatively complete biosynthesis pathway map of phenylpropanoids, flavonoids, and proanthocyanins in blueberry was constructed. The results of the joint analysis showed that the 228 functional genes and 42 TFs regulated 78 differentially expressed metabolites within the biosynthesis pathway of phenylpropanoids/flavonoids. O2PLS analysis results showed that the key metabolites differentially accumulated in blueberry fruit tissues were albireodelphin, delphinidin 3,5-diglucoside, delphinidin 3-O-rutinoside, and delphinidin 3-O-sophoroside, and 10 structural genes (4 Vc4CLs, 3 VcBZ1s, 1 VcUGT75C1, 1 VcAT, and 1 VcUGAT), 4 transporter genes (1 VcGSTF and 3 VcMATEs), and 10 TFs (1 VcMYB, 2 VcbHLHs, 4 VcWD40s, and 3 VcNACs) exhibited strong correlations with 4 delphinidin glycosides. These findings provide insights into the molecular mechanisms of flavonoid biosynthesis and accumulation in blueberry fruit.

HISS: Snakemake-based workflows for performing SMRT-RenSeq assembly, AgRenSeq and dRenSeq for the discovery of novel plant disease resistance genes.

BACKGROUND: In the ten years since the initial publication of the RenSeq protocol, the method has proved to be a powerful tool for studying disease resistance in plants and providing target genes for breeding programmes. Since the initial publication of the methodology, it has continued to be developed as new technologies have become available and the increased availability of computing power has made new bioinformatic approaches possible. Most recently, this has included the development of a k-mer based association genetics approach, the use of PacBio HiFi data, and graphical genotyping with diagnostic RenSeq. However, there is not yet a unified workflow available and researchers must instead configure approaches from various sources themselves. This makes reproducibility and version control a challenge and limits the ability to perform these analyses to those with bioinformatics expertise. RESULTS: Here we present HISS, consisting of three workflows which take a user from raw RenSeq reads to the identification of candidates for disease resistance genes. These workflows conduct the assembly of enriched HiFi reads from an accession with the resistance phenotype of interest. A panel of accessions both possessing and lacking the resistance are then used in an association genetics approach (AgRenSeq) to identify contigs positively associated with the resistance phenotype. Candidate genes are then identified on these contigs and assessed for their presence or absence in the panel with a graphical genotyping approach that uses dRenSeq. These workflows are implemented via Snakemake, a python-based workflow manager. Software dependencies are either shipped with the release or handled with conda. All code is freely available and is distributed under the GNU GPL-3.0 license. CONCLUSIONS: HISS provides a user-friendly, portable, and easily customised approach for identifying novel disease resistance genes in plants. It is easily installed with all dependencies handled internally or shipped with the release and represents a significant improvement in the ease of use of these bioinformatics analyses.

The gene space of European mistletoe (Viscum album).

European mistletoe (Viscum album) is a hemiparasitic flowering plant that is known for its very special life cycle and extraordinary biochemical properties. Particularly, V. album has an unusual mode of cellular respiration that takes place in the absence of mitochondrial complex I. However, insights into the molecular biology of V. album so far are very limited. Since the genome of V. album is extremely large (estimated 600 times larger than the genome of the model plant Arabidopsis thaliana) it has not been sequenced up to now. We here report sequencing of the V. album gene space (defined as the space including and surrounding genic regions, encompassing coding as well as 5' and 3' non-coding regions). mRNA fractions were isolated from different V. album organs harvested in summer or winter and were analyzed via single-molecule real-time sequencing. We determined sequences of 39 092 distinct open reading frames encoding 32 064 V. album proteins (designated V. album protein space). Our data give new insights into the metabolism and molecular biology of V. album, including the biosynthesis of lectins and viscotoxins. The benefits of the V. album gene space information are demonstrated by re-evaluating mass spectrometry-based data of the V. album mitochondrial proteome, which previously had been evaluated using the A. thaliana genome sequence. Our re-examination allowed the additional identification of nearly 200 mitochondrial proteins, including four proteins related to complex I, which all have a secondary function not related to respiratory electron transport. The V. album gene space sequences are available at the NCBI.

A chromosome scale tomato genome built from complementary PacBio and Nanopore sequences alone reveals extensive linkage drag during breeding.

The assembly and scaffolding of plant crop genomes facilitate the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato (Solanum lycopersicum) has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of tobacco mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT Nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We show specific examples of how HiFi and ONT datasets can complement one another to improve assembly contiguity. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all 12 chromosomes were represented as 12 contiguous sequences (N50 = 68.5 Mbp). This chromosome scale assembly did not require scaffolding using an orthogonal data type. The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato genome assemblies that made use of genetic maps and Hi-C for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1-Mbp region of the S. peruvianum genome during tomato breeding. Through marker studies and ONT-based comprehensive haplotyping we show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near-complete genome sequences.

Full-length transcriptome characterization and comparative analysis of Gleditsia sinensis.

As an economically important tree, Gleditsia sinensis Lam. is widely planted. A lack of background genetic information on G. sinensis hinders molecular breeding. Based on PacBio single-molecule real-time (SMRT) sequencing and analysis of G. sinensis, a total of 95,183 non-redundant transcript sequences were obtained, of which 93,668 contained complete open reading frames (ORFs), 2,858 were long non-coding RNAs (LncRNAs) and 18,855 alternative splicing (AS) events were identified. Genes orthologous to different Gleditsia species pairs were identified, stress-related genes had been positively selected during the evolution. AGA, AGG, and CCA were identified as the universal optimal codon in the genus of Gleditsia. EIF5A was selected as a suitable fluorescent quantitative reference gene. 315 Cytochrome P450 monooxygenases (CYP450s) and 147 uridine diphosphate (UDP)-glycosyltransferases (UGTs) were recognized through the PacBio SMRT transcriptome. Randomized selection of GsIAA14 for cloning verified the reliability of the PacBio SMRT transcriptome assembly sequence. In conclusion, the research data lay the foundation for further analysis of the evolutionary mechanism and molecular breeding of Gleditsia.

Stochastic nuclear organization and host-dependent allele contribution in Rhizophagus irregularis.

BACKGROUND: Arbuscular mycorrhizal (AM) fungi are arguably the most important symbionts of plants, offering a range of benefits to their hosts. However, the provisioning of these benefits does not appear to be uniform among AM fungal individuals, with genetic variation between fungal symbionts having a substantial impact on plant performance. Interestingly, genetic variation has also been reported within fungal individuals, which contain millions of haploid nuclei sharing a common cytoplasm. In the model AM fungus, Rhizophagus irregularis, several isolates have been reported to be dikaryotes, containing two genetically distinct types of nuclei recognized based on their mating-type (MAT) locus identity. However, their extremely coenocytic nature and lack of a known single nucleus stage has raised questions on the origin, distribution and dynamics of this genetic variation. RESULTS: Here we performed DNA and RNA sequencing at the mycelial individual, single spore and single nucleus levels to gain insight into the dynamic genetic make-up of the dikaryote-like R. irregularis C3 isolate and the effect of different host plants on its genetic variation. Our analyses reveal that parallel spore and root culture batches can have widely variable ratios of two main genotypes in C3. Additionally, numerous polymorphisms were found with frequencies that deviated significantly from the general genotype ratio, indicating a diverse population of slightly different nucleotypes. Changing host plants did not show consistent host effects on nucleotype ratio's after multiple rounds of subculturing. Instead, we found a major effect of host plant-identity on allele-specific expression in C3. CONCLUSION: Our analyses indicate a highly dynamic/variable genetic organization in different isolates of R. irregularis. Seemingly random fluctuations in nucleotype ratio's upon spore formation, recombination events, high variability of non-tandemly repeated rDNA sequences and host-dependent allele expression all add levels of variation that may contribute to the evolutionary success of these widespread symbionts.

Integrated single-molecule real-time sequencing and RNA sequencing reveal the molecular mechanisms of salt tolerance in a novel synthesized polyploid genetic bridge between maize and its wild relatives.

BACKGROUND: Tripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent in modern maize, especially salt tolerance. A previously reported allopolyploid (hereafter referred to as MTP, 2n = 74) synthesized using Zea mays, Tripsacum dactyloides, and Zea perennis has even stronger salt tolerance than Z. perennis and T. dactyloides. This allopolyploid will be a powerful genetic bridge for the genetic improvement of maize. However, the molecular mechanisms underlying its salt tolerance, as well as the key genes involved in regulating its salt tolerance, remain unclear. RESULTS: Single-molecule real-time sequencing and RNA sequencing were used to identify the genes involved in salt tolerance and reveal the underlying molecular mechanisms. Based on the SMRT-seq results, we obtained 227,375 reference unigenes with an average length of 2300 bp; most of the unigenes were annotated to Z. mays sequences (76.5%) in the NR database. Moreover, a total of 484 and 1053 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Functional enrichment analysis of DEGs revealed that multiple pathways responded to salt stress, including "Flavonoid biosynthesis," "Oxidoreductase activity," and "Plant hormone signal transduction" in the leaves and roots, and "Iron ion binding," "Acetyl-CoA carboxylase activity," and "Serine-type carboxypeptidase activity" in the roots. Transcription factors, such as those in the WRKY, B3-ARF, and bHLH families, and cytokinin negatively regulators negatively regulated the salt stress response. According to the results of the short time series-expression miner analysis, proteins involved in "Spliceosome" and "MAPK signal pathway" dynamically responded to salt stress as salinity changed. Protein-protein interaction analysis revealed that heat shock proteins play a role in the large interaction network regulating salt tolerance. CONCLUSIONS: Our results reveal the molecular mechanism underlying the regulation of MTP in the response to salt stress and abundant salt-tolerance-related unigenes. These findings will aid the retrieval of lost alleles in modern maize and provide a new approach for using T. dactyloides and Z. perennis to improve maize.

PacBio single-molecule long-read sequencing provides new insights into the complexity of full-length transcripts in oriental river prawn, macrobrachium nipponense.

BACKGROUND: Oriental river prawn (Macrobrachium nipponense) is one of the most dominant species in shrimp farming in China, which is a rich source of protein and contributes to a significant impact on the quality of human life. Thus, more complete and accurate annotation of gene models are important for the breeding research of oriental river prawn. RESULTS: A full-length transcriptome of oriental river prawn muscle was obtained using the PacBio Sequel platform. Then, 37.99 Gb of subreads were sequenced, including 584,498 circular consensus sequences, among which 512,216 were full length non-chimeric sequences. After Illumina-based correction of long PacBio reads, 6,599 error-corrected isoforms were identified. Transcriptome structural analysis revealed 2,263 and 2,555 alternative splicing (AS) events and alternative polyadenylation (APA) sites, respectively. In total, 620 novel genes (NGs), 197 putative transcription factors (TFs), and 291 novel long non-coding RNAs (lncRNAs) were identified. CONCLUSIONS: In summary, this study offers novel insights into the transcriptome complexity and diversity of this prawn species, and provides valuable information for understanding the genomic structure and improving the draft genome annotation of oriental river prawn.

High accuracy of single-molecule real-time sequencing in detecting a rare α-globin fusion gene in carrier screening population.

INTRODUCTION: The α-globin fusion gene between the HBA2 and HBAP1 genes becomes clinically important in thalassemia screening because this fusion gene can cause severe hemoglobin (Hb) H disease when combining with α0 -thalassemia (α0 -thal). Due to its uncommon rearrangement in the α gene cluster without dosage changes, this fusion gene is undetectable by common molecular testing approaches used for α-thal diagnosis. METHODS: In this study, we used the single-molecule real-time (SMRT) sequencing technique to detect this fusion gene in 23 carriers identified by next-generation sequencing (NGS) among 16,504 screened individuals. Five primers for α and ß thalassemia were utilized. RESULTS: According to the NGS results, the 23 carriers include 14 pure heterozygotes, eight compound heterozygotes with common α-thal alleles, and one homozygote. By using SMRT, the fusion mutant was successfully detected in all 23 carriers. Furthermore, SMRT corrected the diagnosis in two "pure" heterozygotes: one was compound heterozygote with anti-3.7 triplication, and the other was homozygote. CONCLUSION: Our results indicate that SMRT is a superior method compared to NGS in detecting the α fusion gene, attributing to its efficient, accurate, and one-step properties.

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

BACKGROUND: Trachycarpus 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. RESULTS: A 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. CONCLUSIONS: These 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.