Single-molecule Real-time Sequencing Research Articles

Splicing inhibition mediated by reduced splicing factors and helicases is associated with the cellular response of lung cancer cells to cisplatin

Lung cancer’s mortality is predominantly linked to post-chemotherapy recurrence, driven by the reactivation of dormant cancer cells. Despite the critical role of these reactivated cells in cancer recurrence and metastasis, the molecular mechanisms governing their therapeutic selection remain poorly understood. In this study, we conducted an integrative analysis by combining PacBio single molecule real-time (SMRT) sequencing with short reads Illumina RNA-seq. Our study revealed that cisplatin-induced dormant and reactivated cancer cells exhibited a noteworthy reduction in gene transcripts and alternative splicing events. Particularly, the differential alternative splicing events were found to be overlapping with the differentially expression genes and enriched in genes related to cell cycle and cell division. Utilizing ENCORI database and correlation analysis, we identified key splicing factors, including SRSF7, SRSF3, PRPF8, and HNRNPC, as well as RNA helicase such as EIF4A3, DDX39A, DDX11, and BRIP1, which were associated with the observed reduction in alternative splicing and subsequent decrease in gene expression. Our study demonstrated that lung cancer cells reduce gene transcripts through deminished alternative splicing events mediated by specific splicing factors and RNA helicase in response to the chemotherapeutic stress. These findings provide insights into the molecular mechanisms underlying the therapeutic selection and reactivation of dormant cancer cells. This discovery opens a potential avenue for the development of therapeutic strategies aimed at preventing cancer recurrence following chemotherapy.

Robust Bisulfite‐Free Single‐Molecule Real‐Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme

In addition to the four canonical nucleosides dA, dG, dC and T, genomic DNA contains the additional base 5‐methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter regions or gene bodies significantly affects the transcriptional activity of the corresponding gene. Consequently, the methylation patterns of genes are crucial for either silencing or activating genes. Sequencing the positions of mdC in the genome is therefore of paramount importance for early cancer diagnostics as it helps determine incorrect gene expression. Currently, the bisulfite method is the gold standard for mdC‐sequencing. However, this method has the drawback that the majority of the input DNA is degraded during the bisulfite treatment. Additionally, bisulfite sequencing is prone to errors. Here, we report a benign, bisulfite‐free mdC sequencing method termed EMox‐seq, which is based on third‐generation single‐molecule SMRT sequencing. The foundation of this technology is a new Tet3 enzyme that efficiently oxidizes mdCs to 5‐carboxycytidine (cadC). In turn, cadC provides an excellent readout by SMRT sequencing using specially trained AI‐based algorithms.

Robust Bisulfite-Free Single-Molecule Real-Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme.

In addition to the four canonical nucleosides dA, dG, dC and T, genomic DNA contains the additional base 5-methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter regions or gene bodies significantly affects the transcriptional activity of the corresponding gene. Consequently, the methylation patterns of genes are crucial for either silencing or activating genes. Sequencing the positions of mdC in the genome is therefore of paramount importance for early cancer diagnostics as it helps determine incorrect gene expression. Currently, the bisulfite method is the gold standard for mdC-sequencing. However, this method has the drawback that the majority of the input DNA is degraded during the bisulfite treatment. Additionally, bisulfite sequencing is prone to errors. Here, we report a benign, bisulfite-free mdC sequencing method termed EMox-seq, which is based on third-generation single-molecule SMRT sequencing. The foundation of this technology is a new Tet3 enzyme that efficiently oxidizes mdCs to 5-carboxycytidine (cadC). In turn, cadC provides an excellent readout by SMRT sequencing using specially trained AI-based algorithms.

Characterization of a plant‐pathogenic T3SS‐lacking Xanthomonas strain isolated from common ragweed

AbstractCommon ragweed (Ambrosia artemisiifolia) is one of the leading causes of allergenic rhinitis, as well as a major weed of many crops. Biological control with plant pathogens, such as fungi and bacteria, represents an attractive alternative to the application of synthetic herbicides to control this noxious weed. In this study, we isolated a Xanthomonas strain (designated as 10‐10) from a diseased common ragweed plant collected in southern Quebec, Canada. We characterized the bioherbicidal potential of this strain against common ragweed, and determined whether it can infect other plant species. Its genome was sequenced using PacBio's SMRT technology, enabling us to gain insight into the phylogenetic placement of this strain within the genus Xanthomonas, and to study the repertoire of virulence‐associated genes and clusters. Xanthomonas sp. 10‐10 belongs to a clearly defined subclade within clade C (group 2), which includes X. vesicatoria, X. dyei and X. pisi. While this strain is closely related to X. pisi, it represents a new species within the genus Xanthomonas. Surprisingly, this strain lacks a type III secretion system, which is the main virulence determinant in pathogenic Xanthomonas spp., but harbours an arsenal of cell wall‐degrading enzymes. The addition of the organosilicone surfactant Silwet L‐77 drastically increased the disease symptoms caused by Xanthomonas sp. 10‐10 on common ragweed plants grown under controlled conditions, but the plants remained alive. This strain also caused disease symptoms on tomato, pepper and lettuce. More research is needed to develop Xanthomonas‐based bioherbicide specifically targeting common ragweed.

Genomic insights into bamboo witches’ broom disease: pathogenicity and phytohormone biosynthesis in Aciculosporium take

Bamboo witches’ broom disease (WBD), caused by Aciculosporium take Miyake, devastates bamboo forests. Understanding the genome and pathogenic factors of pathogen is crucial for disease control. We employed single-molecule real-time sequencing, Illumina paired-end sequencing, and chromatin interaction mapping techniques to assemble the genome of A. take CCTCC-M2023413, analyze pathogenicity- and phytohormone-biosynthesis-related genes, and compare it to 12 other WBD pathogens. The genome of A. take is 59.24 Mb in size, with 54.32% repeats, 7 chromosomes, 7,105 protein-coding genes, 84 ribosomal RNAs, and 115 transfer RNAs. Predictive analysis of pathogenicity genes found 237 carbohydrate-active enzymes, 1,069 membrane transport proteins, 1,040 pathogen-host interaction genes, 315 virulence factors, and 70 effectors. Most of pathogenicity genes overlapped with repeat-rich regions. Additionally, 172 genes were linked to auxin biosynthesis, 53 to brassinosteroid biosynthesis, and 2 to cis-zeatin biosynthesis. Comparative genomic analysis identified 77 core orthogroups shared by 13 WBD pathogens, played roles in metabolites, genetic information processing, pathogenesis, cis-zeatin biosynthesis, lifespan, and quorum sensing. The miaA gene, crucial for cis-zeatin biosynthesis, is structurally conserved and sequence-diverse among 13 WBD pathogens, with upregulated expression during bamboo WBD pathogenesis. This highlights that cis-zeatin is significant contributor to host pathogenesis, and miaA is a new potential target for controlling WBD. This study provides important insights on preventing and controlling bamboo WBD.

Metasilicate-based alkaline mineral water improves the growth performance of weaned piglets by maintaining gut-liver axis homeostasis through microbiota-mediated secondary bile acid pathway

Weaning stress causes substantial economic loss in the swine industry. Moreover, weaning-induced intestinal barrier damage and dysfunction of the gut-liver axis are associated with reduced growth performance in piglets. Metasilicate-based alkaline mineral water (AMW) has shown potential therapeutic effects on gastrointestinal disorders; however, the mechanisms involved and their overall effects on the gut-liver axis have not been explored. Here, sodium metasilicate (SMS) was used to prepare metasilicate-based AMW (basal water + 500 mg/L SMS). A total of 240 newly weaned piglets were allocated to the Control and SMS groups (6 replicate pens per group and 20 piglets per pen) for a 15-day trial period. Histopathological evaluations were conducted using hematoxylin and eosin staining. To analyze the composition of the gut microbiota, 16S rRNA PacBio SMRT Gene Full-Length Sequencing was performed. Western blotting and immunofluorescence were employed to assess protein expression levels. Our results indicated that metasilicate-based AMW effectively alleviated weaning-induced colonic or liver morphological injury and inflammatory response, as well as liver cholesterol metabolism disorders. Further analysis showed that metasilicate-based AMW promoted deoxycholic acid (DCA) biosynthesis by increasing the abundance of Lactobacillus_delbrueckii in the colon (P < 0.001). This consequently improved weaning-induced colon and liver injury and dysfunction through the DCA-secondary bile acid (SBA) receptors (SBAR)-nuclear factor-kappaB (NF-κB)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) pathways. Growth performance parameters, including final body weight (P = 0.034) and average daily gain (P < 0.001), in the SMS group were significantly higher than those in the Control group. Therefore, metasilicate-based AMW maintains gut-liver axis homeostasis by regulating the microbiota-mediated SBA-SBAR pathway in piglets under weaning stress. Our research provides a new strategy for mitigating stress-induced gut-liver axis dysfunction in weaned piglets.

Application of high-throughput sequencing in research on plant endophytes

Plant endophytes spend at least part of their life cycle in plants without causing diseases in the hosts, being the microbial resources with rich species and diverse functions. With the advancement in sequencing technology, the microbiological study of endophytes has become increasingly intensive. Being praised for the targeted validation and low cost, Sanger sequencing has been preferred by researchers. However, Sanger sequencing is no longer suitable for deeper genomic study of endophytes due to the low throughput. In this paper, we briefly summarize the research history of endophytes, review the applications of next-generation sequencing characterized by high throughput and third-generation sequencing (single-molecule real-time sequencing) in the research on endophytes, and summarize the research results of different sequencing technologies. Furthermore, we summarize the advantages and limitations of different sequencing technologies and discuss how to choose the appropriate sequencing technology according to the research needs. This review provides a reference for researchers to further explore the potential value of plant endophytes.

Genome sequence of an extremely halophilic archaeon isolated from Permian Period halite, Salado Formation in New Mexico, USA: Halobacterium sp. strain NMX12-1.

Halobacterium sp. strain NMX12-1, an extremely halophilic Archaeon, was isolated from 250 million-year-old Salado Formation salt crystal in Carlsbad, New Mexico. Single-molecule real-time sequencing revealed a 3.2-Mbp genome with a 2.6-Mbp chromosome and five plasmids (234, 211, 119, 21, and 1.6-kbp). The GC-rich genome encodes an acidic proteome, characteristic of Haloarchaea.

Combining full-length transcriptome sequencing and next generation sequencing to provide insight into the growth superiority of the hybrid grouper (Cromileptes altivelas (♀) × Epinephelus lanceolatus (♂)).

The hybrid grouper (Cromileptes altivelas, ♀ × Epinephelus lanceolatus, ♂) is an economically important aquaculture species that exhibits certain growth advantages compared to its female parent, Cromileptes altivelas. However, the current understanding of the molecular mechanisms underlying the growth of hybrid groupers is lacking. Herein, we performed full-length transcriptome sequencing and next-generation sequencing on the hybrid grouper and its parents to identify growth-related genes and comprehensively analyze the regulatory mechanism of growth heterosis in the hybrid grouper. Approximately 44.70, 40.44, and 45.32 Gb of single-molecule real-time sequencing data were generated in C. altivelas (Cal), E. lanceolatus (Ela), and the hybrid (Hyb), which were combined into 204,322 non-redundant isoforms using the PacBio sequencing platform. Differentially expressed genes (DEGs) were identified between Hyb and Cal (3,494, 2,125, and 1,487 in brain, liver, and muscle tissues, respectively) and Hyb and Ela (3,415, 2,351, and 1,675 in brain, liver, and muscle tissues, respectively). Then, 27 DEGs (13 in the brain and 14 in the muscle) related to growth traits were identified using cluster and correlation network analysis. Quantitative RT-PCR validated 15 DEGs consistent with transcriptome sequencing (RNA-seq) trends. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these 15 genes were mainly involved in regulating the actin cytoskeleton, suggesting that this pathway plays an essential role in fish growth. In addition, we found that the phosphatase and tensin homologue (PTEN) is a key regulator of growth heterosis in Hyb. These results shed light on the regulatory mechanism of growth in the Hyb, which is important for marker-assisted selection programs to improve the growth quality of groupers.

Integrated PacBio SMRT and Illumina sequencing uncovers transcriptional and physiological responses to drought stress in whole-plant Nitraria tangutorum.

Nitraria tangutorum Bobr., a prominent xerophytic shrub, exhibits remarkable adaptability to harsh environment and plays a significant part in preventing desertification in northwest China owing to its exceptional drought and salinity tolerance. To investigate the drought-resistant mechanism underlying N. tangutorum, we treated 8-week-old seedlings with polyethylene glycol (PEG)-6000 (20%, m/m) to induce drought stress. 27 samples from different tissues (leaves, roots and stems) of N. tangutorum at 0, 6 and 24h after drought stress treatment were sequenced using PacBio single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing to obtain a comprehensive transcriptome. The PacBio SMRT sequencing generated 44,829 non-redundant transcripts and provided valuable reference gene information. In leaves, roots and stems, we identified 1162, 2024 and 232 differentially expressed genes (DEGs), respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that plant hormone signaling and mitogen-activated protein kinase (MAPK) cascade played a pivotal role in transmitting stress signals throughout the whole N. tangutorum plant following drought stress. The interconversion of starch and sucrose, as well as the biosynthesis of amino acid and lignin, may represent adaptive strategies employed by N. tangutorum to effectively cope with drought. Transcription factor analysis showed that AP2/ERF-ERF, WRKY, bHLH, NAC and MYB families were mainly involved in the regulation of drought response genes. Furthermore, eight physiological indexes, including content of proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), total amino acid and soluble sugar, and activities of three antioxidant enzymes were all investigate after PEG treatment, elucidating the drought tolerance mechanism from physiological perspective. The weighted gene co-expression network analysis (WGCNA) identified several hub genes serve as key regulator in response to drought through hormone participation, ROS cleavage, glycolysis, TF regulation in N. tangutorum. These findings enlarge genomic resources and facilitate research in the discovery of novel genes research in N. tangutorum, thereby establishing a foundation for investigating the drought resistance mechanism of xerophyte.

Comparison of short-read and long-read next-generation sequencing technologies for determining HIV-1 drug resistance.

Accurate HIV-1 genome sequencing is necessary to identify drug resistance mutations (DRMs) in people with HIV-1 (PWH). Next-generation-sequencing (NGS) allows the detection of minor variants and is now available in many laboratories. Our study aimed to compare two NGS approaches, a "short read" sequencing protocol using DeepChek® Whole Genome HIV-1 Assay on Illumina, and a "long read" sequencing protocol of HIV-1 pol and env single-molecule real-time sequencing (SMRT) on Pacific Biosciences (PacBio). We analyzed 16 plasma samples and 13 cellular samples from PWH. HIV-1 whole genome was amplified into five amplicons using DeepChek® Whole Genome HIV-1 Assay and sequenced on an iSeq. 100. In parallel, HIV-1 pol and env genes were separately amplified and sequenced using PacBio SMRT system with the circular consensus sequencing mode on a Sequel IIe. Concordance rates for determining DRMs with both approaches varied depending on the HIV-1 region, with higher concordance in the integrase region compared to the reverse transcriptase and protease regions. DeepChek® Whole Genome HIV-1 Assay exhibited better sensitivity in HIV-1 RNA sequencing of plasmas with lower viral loads. In cell HIV-1 DNA sequencing, the DeepChek® Whole Genome HIV-1 Assay performed better in pol and env sequencing but detected more APOBEC-induced DRMs, which can represent defective proviruses. Our findings indicate that both DeepChek® Whole Genome HIV-1 Assay and PacBio SMRT sequencing exhibit good performance for subtype determination, detection, and quantification of DRMs of the HIV-1 genome. However, some discrepancies were found in cellular samples, highlighting the challenges of interpreting HIV-1 DNA DRMs.

Bacterial and fungal aerosols in poultry houses: PM2.5 metagenomics via single-molecule real-time sequencing

Microbial aerosol contamination is a common problem in poultry farms, posing potential health risks to poultry and their caretakers. Exploring the distribution and diversity of the microbial community in poultry farm aerosols is crucial for effective mitigation strategies. The composition of bacterial and fungal aerosols is poorly understood, particularly the prevalence of potential pathogenic microorganisms in fine particulate matter (PM2.5) in various types of poultry houses. In this study, 27 PM2.5 samples were collected from 5 chicken houses and 4 duck houses in Shandong Province, China. Species-level diversity of bacterial and fungal components in PM2.5 samples was determined using advanced single-molecule real-time sequencing (SMRT) technology, based on the 16S and internal transcribed spacer 1 (ITS) ribosomal genes. Microbial diversity and community composition varied significantly across the different poultry house. Notably, duck houses had higher concentrations (p < 0.01) of PM2.5 (92.8-143.1 μg/m3) than chicken houses (42.0-56.4 μg/m3). Furthermore, microbial variation in PM2.5 samples was associated with the type of poultry facility. The predominant pathogenic microorganisms included Aspergillus sydowii, Penicillium sp., Aspergillus insolitus, Cladosporium sp., Aspergillus sp., Aspergillus pseudoglaucus, Cladosporium sp. C4092-2-PD1, and Colletotrichum sp., all of which were classified as second category of pathogens. Aspergillus sydowii and Penicillium sp. were the dominant species in chicken houses, while Cladosporium sp., Aspergillus sp., and Aspergillus pseudoglaucus were the dominant species identified in duck houses. To our knowledge, this study is the first to investigate bacterial and fungal diversity in PM2.5 samples collected from various types of poultry houses. These findings advance our understanding of poultry environmental microbiology and have important implications for safeguarding the health of both poultry and their caretakers.

Chromosome-level genome assembly and annotation of the Spinibarbus caldwelli

Spinibarbus caldwelli is an important freshwater economic fish in China. Owing to uncontrolled fishing, wild resources of S. caldwelli have decreased rapidly and may be on the verge of extinction. In this study, utilizing single-molecule real-time (SMRT) sequencing technology and chromatin interaction mapping (Hi-C) technologies, we assembled the first chromosome-scale genome for S. caldwelli about 1.77 Gb in size, with a contig N50 length of 11.83 Mb and scaffold N50 length of 33.91 Mb. In total 1.72 Gb (97.01%) of the contig sequences were anchored onto fifty chromosomes with the longest scaffold being 56.20 Mb. Furthermore, proximately 49.41% of the genome was composed of repetitive elements. In total, 49,377 protein-coding genes were predicted, of which 47,724 (96.65%) genes have been functionally annotated. The high-quality chromosome-level reference genome and annotation are vital for supporting basic genetic studies and will be contribute to genetic structure, functional elucidation, evolutionary inquiry, and germplasm conservation for S. caldwelli.

Full-Length Transcriptome Construction and Systematic Characterization of Virulence Factor-Associated Isoforms in Vairimorpha (Nosema) Ceranae.

Vairimorpha (Nosema) ceranae is a single-cellular fungus that obligately infects the midgut epithelial cells of adult honeybees, causing bee microsporidiosis and jeopardizing bee health and production. This work aims to construct the full-length transcriptome of V. ceranae and conduct a relevant investigation using PacBio single-molecule real-time (SMRT) sequencing technology. Following PacBio SMRT sequencing, 41,950 circular consensus (CCS) were generated, and 25,068 full-length non-chimeric (FLNC) reads were then detected. After polishing, 4387 high-quality, full-length transcripts were gained. There are 778, 2083, 1202, 1559, 1457, 1232, 1702, and 3896 full-length transcripts that could be annotated to COG, GO, KEGG, KOG, Pfam, Swiss-Prot, eggNOG, and Nr databases, respectively. Additionally, 11 alternative splicing (AS) events occurred in 6 genes were identified, including 1 alternative 5' splice-site and 10 intron retention. The structures of 225 annotated genes in the V. ceranae reference genome were optimized, of which 29 genes were extended at both 5' UTR and 3' UTR, while 90 and 106 genes were, respectively, extended at the 5' UTR as well as 3' UTR. Furthermore, a total of 29 high-confidence lncRNAs were obtained, including 12 sense-lncRNAs, 10 lincRNAs, and 7 antisense-lncRNAs. Taken together, the high-quality, full-length transcriptome of V. ceranae was constructed and annotated, the structures of annotated genes in the V. ceranae reference genome were improved, and abundant new genes, transcripts, and lncRNAs were discovered. Findings from this current work offer a valuable resource and a crucial foundation for molecular and omics research on V. ceranae.

Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Detecting serologically difficult ABO blood groups using single-molecule real-time sequencing technology.

Recently, third-generation long-read sequencing technology has been increasingly applied to the detection of various blood group systems. Because of its long read length and use of single-molecule sequencing, it is capable of obtaining the sequences of blood group genes in their entirety as well as of distinguishing haplotypes. Therefore, here, we collected ABO blood group samples that were difficult to classify serologically and analysed the sequences of the coding regions of the ABO genes as well as the sequences upstream and downstream of the coding regions. Samples with ABO antigen typing and reverse serum typing discrepancies were screened in a total of 21 patients. All samples were subjected to serological testing and preliminary ABO genotyping (polymerase chain reaction with sequence-specific primers [PCR-SSP]), followed by single-molecule real-time (SMRT) sequencing to obtain complete ABO gene sequences. PCR sequence-based typing (PCR-SBT) was performed to validate the results. Of the 21 samples, 15 had common ABO types, and 6 had rare ABO subtypes. One new allele, ABO*B.NEW (c.861C>T), and one allelic base recombination event was identified. Forty-two haplotype sequences were obtained via SMRT sequencing with intronic single-nucleotide variants (SNVs) specific to the ABO allele, and all of the exon region sequences were consistent with the PCR-SBT results. SMRT sequencing is capable of accurately obtaining complete ABO gene sequences, distinguishing haplotypes and identifying allelic recombination.

Draft genome sequence of Penicillium citrinum B9, a plant growth-promoting symbiont from barley rhizosphere.

Penicillium citrinum strain B9 is a plant growth-promoting fungus isolated from Barley (Hordeum vulgare) rhizosphere. We report the first draft genome of P. citrinum B9 assembled using single-molecule real-time sequencing and Illumina reads. The assembled genome spans 31.3 Mb comprising nine contigs and 10,106 protein-encoding genes.

High throughput long-read sequencing of circulating lymphocytes of the evolutionarily distant sea lamprey reveals diversity and common elements of the variable lymphocyte receptor B (VLRB) repertoire.

The leucine-rich repeat-based variable lymphocyte receptor B (VLRB) antibody system of jawless vertebrates is capable of generating an antibody repertoire equal to or exceeding the diversity of antibody repertoires of jawed vertebrates. Unlike immunoglobulin-based immune repertoires, the VLRB repertoire diversity is characterized by variable lengths of VLRB encoding transcripts, rendering conventional immunoreceptor repertoire sequencing approaches unsuitable for VLRB repertoire sequencing. Here we demonstrate that long-read single-molecule real-time (SMRT) sequencing (PacBio) approaches permit the efficient large-scale assessment of the VLRB repertoire. We present a computational pipeline for sequence data processing and provide the first repertoire-based analysis of VLRB protein characteristics including properties of its subunits and regions of diversity within each structural leucine-rich repeat subunit. Our study provides a template to explore changes in the VLRB repertoire during immune responses and to establish large scale VLRB repertoire databases for computational approaches aimed at isolating monoclonal VLRB reagents for biomedical research and clinical applications.

Chromosome‐scale genome assembly of the autoalloenneaploid Arundo donax

AbstractBackgroundArundo donax L. has great potential as an energy crop due to its high biomass yield and broad adaptability, while lack of a reference genome is a hindrance to genetic improvement efforts for this species.MethodsGenome assembly of A. donax was conducted by utilizing PacBio SMRT sequencing and high‐throughput chromosome conformation capture technology, with further analysis exploring the plant's ploidy, whole‐genome duplication event, and evolutionary history through comparative genomics.ResultsThe genome assembly of A. donax consists of 1.30 Gb with a contig N50 size of 33.15 Mb. A total of 74 403 gene models were identified, with over 90% of genes being functionally annotated. Karyotype and synteny analyses revealed that A. donax is an autoalloenneaploid (3n = 9x = 108) and has experienced significant gene family expansion and two whole‐genome duplication events during its evolutionary history. Furthermore, utilizing the genome assembly, a variety of salinity stress‐related genes were uncovered through the analysis of public RNA‐seq data.ConclusionsThis study presents the initial chromosome‐scale genome assembly of A. donax, which will advance genetic comprehension and support the genetic enhancement of this important energy crop.

Identification of 43 novel HLA alleles by PacBio single molecule real-time sequencing in haematopoietic cell donors.

A total of 43 novel HLA alleles detected in haematopoietic cell donors using single molecule real-time DNA sequencing.