Long-read Sequencing Technologies Research Articles

(Invited) BioFETs and Nanopore FETs: Nanoscale Silicon Field-Effect Transistors for Single-Molecule Sensing

High read throughput single-molecule sensing is a cornerstone of established third generation long-read DNA sequencing technologies and is crucial for emerging protein sequencing technologies. These omics technologies are of great interest for essential understanding and applications in the life sciences. Field Effect Transistor (FET)-based single-molecule sensors promise advances in omics, by further enhancing read throughput with massive parallelization.Here an overview is given of our recent progress on nanoscale bioFETs and nanopore FETs (NPFETs).A bioFET is a FET gated by a liquid electrolyte rather than by a solid gate. The FET is turned into a biosensor by attaching probe biomolecules to the gate dielectric. These probes bind specifically to the target biomolecule of interest. The attachment of the targets to the probes on the gate dielectric surface shifts the FET threshold voltage.A nanopore FET is an electrolytically gated FET as well, in which the gate gauges potential changes caused by a molecule moving, or translocating, through a nanopore, a nanoscale opening connecting two reservoirs. Nanopores have been firmly established as single-molecule sensors as the ionic current running through them is modulated by passing single molecules.Nanoscale silicon bioFET sensors have single-molecule capabilities within their reach [1-4]. To realize single-molecule sensing the single-molecule signal-to-noise ratio (SNR) must be sufficiently high. Downscaling the device area is key to increase SNR (∝1/√area) [5]. Although many reported FET sensors have nanoscale cross-sections, their gate lengths are typically micron-scale. Our FET modeling work shows that a sufficiently large single-molecule Signal-to-Noise Ratio (SNR) can be obtained when nano-scaling all FET channel dimensions [6]. We have shown significant progress [1] toward single-molecule detection by fabricating and characterizing finFET sensors with a gate length of 50nm and have shown that Si FETs have the potential to enable single-molecule sensing.Noise plays a central role in single-molecule sensing [7,8] and drift can also be a challenge in FET sensors [9]. Perhaps the greatest challenge for bioFETs is electrolyte screening which reduces the signal by masking the charge of target molecules with counterions. We have experimentally established the importance of non-linear screening, and the pH interference effect which can aggravate screening [10,11] and propose solutions.The nanopore FET, senses single molecules translocating through the pore due to the so-called resistive divider effect [6]. The nanopore FET has been experimentally demonstrated as a single-molecule sensor in 2012 [12]. The nanopore FET’s device-level advantage is its potential high > 100 MHz bandwidth with sufficient signal-to-noise ratio (SNR). This has been studied theoretically [13] but is yet to be proven experimentally. The second, and most important advantage, is the superior integrability of nanopore-FETs into large-scale arrays.In our recent simulation work making use of a dedicated nanofluidic nanodevice simulator we reveal that sensitivity can be boosted exponentially by increasing cis-trans bias [14].We have fabricated and characterized a Nanopore-FET milestone device [15,16], which has a nanopore running through a nanoscale silicon FET channel, but without bottom reservoir (see Fig. 1). On this milestone device we obtain excellent electrolytically gated FET transfer and output characteristics demonstrating a low subthreshold swing of 67mV/decade, indicating excellent device quality.Applications of the nanopore FET for omics and DNA data storage will be discussed.FET-based sensing promises to strongly enhance the read throughput of single-molecule sensing by means of massive parallelization.<ECSTEM.jpg>Fig. 1. TEM cross-section of the NPFET milestone device.[1] Santermans et al. IEEE International Electron Devices Meeting, 2020[2] Li et al. ACS Nano 2017, 11, 12789−12795[3] He et al. Angewandte Chemie, 55, 1, 2016[4] Li et al. Nanoscale, 2016, 8, 16172[5] Männik et al. Nano Letters, 8, 2008[6] Santermans et al., to be published[7] Martens et al. Int. Conf. Noise Fluctuations (ICNF), 2019[8] Bergfeld Mori et al. Solid-State Electronics 194, p. 108358, 2022[9] Wuytens et al. IEEE Int. Reliability Physics Symposium, 2020[10] Gupta, et al. IEEE Sensors Journal, 21, 4, p. 4143, 2020[11] Santermans et al. ACS Sensors 6, 3, p. 1049, 2021[12] Xie et al. Nature Nanotechnology, 7(2), pp.119-125, 2012[13] Parkin and Drndić, ACS sensors, 3, 2, p. 313, 2018[14] Verhulst et al., IEEE Sensors Journal, 22, 6, pp.5732-5742, 2022[15] Liu et al., MNE, Leuven, Sep., 2022[16] Martens et al. IEEE International Electron Devices Meeting, 2022 Figure 1

Comparing assembly strategies for third-generation sequencing technologies across different genomes

The recent advent of long-read sequencing technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore technology (ONT), has led to substantial accuracy and computational cost improvements. However, de novo whole-genome assembly still presents significant challenges related to the computational cost and the quality of the results. Accordingly, sequencing accuracy and throughput continue to improve, and many tools are constantly emerging. Therefore, selecting the correct sequencing platform, the proper sequencing depth and the assembly tools are necessary to perform high-quality assembly. This paper evaluates the primary assembly reconstruction from recent hybrid and non-hybrid pipelines on different genomes. We find that using PacBio high-fidelity long-read (HiFi) plays an essential role in haplotype construction with respect to ONT reads. However, we observe a substantial improvement in the correctness of the assembly from high-fidelity ONT datasets and combining it with HiFi or short-reads.

AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology

AbstractAcute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

Resolving complex structural variants via nanopore sequencing.

The recent development of high-throughput sequencing platforms provided impressive insights into the field of human genetics and contributed to considering structural variants (SVs) as the hallmark of genome instability, leading to the establishment of several pathologic conditions, including neoplasia and neurodegenerative and cognitive disorders. While SV detection is addressed by next-generation sequencing (NGS) technologies, the introduction of more recent long-read sequencing technologies have already been proven to be invaluable in overcoming the inaccuracy and limitations of NGS technologies when applied to resolve wide and structurally complex SVs due to the short length (100-500bp) of the sequencing read utilized. Among the long-read sequencing technologies, Oxford Nanopore Technologies developed a sequencing platform based on a protein nanopore that allows the sequencing of "native" long DNA molecules of virtually unlimited length (typical range 1-100Kb). In this review, we focus on the bioinformatics methods that improve the identification and genotyping of known and novel SVs to investigate human pathological conditions, discussing the possibility of introducing nanopore sequencing technology into routine diagnostics.

The characterisation of antimicrobial resistant Escherichia coli from dairy calves.

Introduction. Dairy calves, particularly pre-weaned calves have been identified as a common source of multidrug resistant (MDR) Escherichia coli.Gap statement. E. coli strains isolated from dairy calves and the location of their resistance genes (plasmid or chromosomal) have not been well characterised.Aim. To characterise the phenotypic and genotypic features as well as the population structure of antimicrobial-resistant E. coli isolated from calves located on dairy farms that feed waste-milk to their replacement calves.Methodology. Recto-anal swab enrichments from 40 dairy calves (≤ 14 days old) located on four dairy farms were examined for tetracycline, streptomycin, ciprofloxacin, and third-generation cephalosporin resistant E. coli. Whole genome sequencing was performed using both short- and long-read technologies on selected antimicrobial resistant E. coli.Results. Fifty-eight percent (23/40) of calves harboured antimicrobial resistant E. coli: 43 % (17/40) harboured tetracycline resistant, and 23 % (9/40) harboured chromosomal mediated AmpC producing E. coli. Whole genome sequencing of 27 isolates revealed five sequence types, with ST88 being the dominant ST (17/27, 63 % of the sequenced isolates) followed by ST1308 (3/27, 11 %), along with the extraintestinal pathogenic E. coli lineages ST69 (3/27, 11 %), ST10 (2/27, 7 %), and ST58 (2/27, 7 %). Additionally, 16 isolates were MDR, harbouring additional resistance genes that were not tested phenotypically. Oxford Nanopore long-read sequencing technologies enabled the location of multiple resistant gene cassettes in IncF plasmids to be determined.Conclusion. Our study identified a high incidence of tetracycline and streptomycin-resistant E. coli in dairy calves, and highlighted the presence of multidrug-resistant strains, emphasising the need for further investigation into potential associations with farm management practices.

De novo genome assembly and comparative genomics for the colonial ascidian Botrylloides violaceus.

Ascidians have the potential to reveal fundamental biological insights related to coloniality, regeneration, immune function, and the evolution of these traits. This study implements a hybrid assembly technique to produce a genome assembly and annotation for the botryllid ascidian, Botrylloides violaceus. A hybrid genome assembly was produced using Illumina, Inc. short and Oxford Nanopore Technologies long-read sequencing technologies. The resulting assembly is comprised of 831 contigs, has a total length of 121 Mbp, N50 of 1 Mbp, and a BUSCO score of 96.1%. Genome annotation identified 13 K protein-coding genes. Comparative genomic analysis with other tunicates reveals patterns of conservation and divergence within orthologous gene families even among closely related species. Characterization of the Wnt gene family, encoding signaling ligands involved in development and regeneration, reveals conserved patterns of subfamily presence and gene copy number among botryllids. This supports the use of genomic data from nonmodel organisms in the investigation of biological phenomena.

Ribonucleic Acid-Mediated Control of Protein Translation Under Stress.

Significance: The need of cells to constantly respond to endogenous and exogenous stress has necessitated the evolution of pathways to counter the deleterious effects of stress and to restore cellular homeostasis. The inability to activate a timely and adequate response can lead to disease and is a hallmark of aging. Besides protein-coding genes, cells contain a plethora of noncoding regulatory elements that allow cells to respond rapidly and efficiently to external stimuli by activating highly specific and tightly controlled mechanisms. Many of these programs converge on the regulation of translation, one of the most energy-consuming processes in cells. Recent Advances: The noncoding dimension of translational regulation includes short and long noncoding ribonucleic acids (ncRNAs), as well as messenger RNA features, such as the sequence and modification status of the 5' and 3' untranslated regions (UTRs), that do not change the amino acid sequence of the produced protein. Critical Issues: In this review, we discuss the regulatory role of the nonprotein-coding components of translation under stress, particularly oxidative stress. We conclude that the regulation of translation through ncRNAs, UTRs, and nucleotide modifications is emerging as a critical component of the stress response. Future Directions: Further areas of study using long-read sequencing technologies will be discussed. Antioxid. Redox Signal. 39, 374-389.

Single-cell third-generation sequencing-based multi-omics uncovers gene expression changes governed by ecDNA and structural variants in cancer cells.

Cancer cells often exhibit large-scale genomic variations, such as circular extrachromosomal DNA (ecDNA) and structural variants (SVs), which have been highly correlated with the initiation and progression of cancer. Currently, no adequate method exists to unveil how these variations regulate gene expression in heterogeneous cancer cell populations at a single-cell resolution. Here, we developed a single-cell multi-omics sequencing method, scGTP-seq, to analyse ecDNA and SVs using long-read sequencing technologies. We demonstrated that our method can efficiently detect ecDNA and SVs and illustrated how these variations affect transcriptomic changes in various cell lines. Finally, we applied and validated this method in a clinical sample of hepatocellular carcinoma (HCC), demonstrating a feasible way to monitor the evolution of ecDNA and SVs during cancer progression.

A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses.

Bacteriophages (phages) within the genus Przondovirus are T7-like podoviruses belonging to the subfamily Studiervirinae, within the family Autographiviridae, and have a highly conserved genome organisation. The genomes of these phages range from 37 to 42 kb in size, encode 50-60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA, a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our assembly workflow utilised Oxford Nanopore Technologies for long-read sequencing for its accessibility, making it the more relevant long-read sequencing technology at this time, and Illumina DNA Prep for short-read sequencing, representing the most commonly used technologies globally. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics.

Determination of Whole Genome Sequence and Resistance Genes in Hypermucoviscous Klebsiella pneumoniae with Long-Read Sequencing Technology

Numerous virulence factors are present in the hypervirulent/hypermucoviscous Klebsiella pneumoniae, which primarily causes community-acquired infections. In recent years, hypervirulent/hypermucoviscous K.pneumoniae has acquired resistance genes and has been linked to healthcare-associated infections. The aims of this study were to determine whole genome sequencing of hypermucoviscous K.pneumoniae that induces healthcare-associated bloodstream infection utilizing the Oxford Nanopore (MinION) platform, to identify resistance genes using various databases, and to compare the database results. K.pneumoniae isolates that were sent to the Dokuz Eylül University Research and Training Hospital Center Laboratory and were isolated from blood cultures were included in this study between January 2018 and December 2020. K.pneumoniae isolates were identified using the automated VITEK-2 system. The disc diffusion method was used to characterize the antimicrobial resistance profile, and the string test was used to assess the hypermucoviscous phenotype. By using specific primers blaOXA-48, blaNDM, blaKPC, blaIMP, and blaVIM, carbapenem resistance genes were investigated using the PCR method. To ascertain clonal relatedness among hypermucoviscous K.pneumoniae isolates, PFGE was used. The whole genome sequencing of five K.pneumoniae strains with different origins was determined by Oxford Nanopore (MinION) technology. Using the ResFinder, CARD and BacWGSTdb databases, resistance genes were examined. Capsule regulation genes were analyzed with the BacWGSTdb database. Resistance genes on the plasmid were discovered using the ResFinder database after plasmid analyses were carried out using the PLSDB and PlasmidFinder databases. A total of 244 K.pneumoniae isolates were included in the study. Ten isolates were found to be hypermucoviscous. A carbapenem-resistant hypermucoviscous K.pneumoniae isolate was discovered to carry the blaOXA-48. Five hypermucoviscous isolates of which whole genome sequences were determined had blaSHV types, oqxB, oqxA, and fosA genes on the chromosome. Capsule regulation genes rcsA, rcsB were detected in all five isolates, and rmpA/rmpA2 genes caused hypermucoviscous phenotype was detected in two of the five isolates. In the plasmid analysis, IncFIB (K) pCAV1099- 14, Col (pHAD28), IncFIA (HI1)-IncR, Col (pHAD28), Col440, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR), IncL, IncFIB (K)-IncHI1 (K), IncFIB (K), IncR, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR)-IncR, repB plasmids were identified. Resistance genes; aac(3)-IId, aph(6)-Id, aph(3")-Ib qnrB1, sul2, dfrA14, blaTEM-1A, blaCTX-M-15, armA, msr(E), mph(E), catB3, blaOXA-1, aac(6')-Ib-cr, catA1 and blaOXA-48 were detected on the plasmid. In the bioinformatic analyzes, it was determined that two study isolates with hypermucoviscous phenotype had various plasmids and carried many resistance genes on these plasmids. Various resistance and virulence genes are spread through plasmids and the number of resistant hypermucoviscous K.pneumoniae is increasing day by day. Appropriate infection control strategies should be implemented to prevent healthcare-associated infections and outbreaks caused by antimicrobial-resistant hypermucoviscous K.pneumoniae. For this purpose, resistance monitoring of antimicrobials used in the treatment of K.pneumoniae infections should be performed. There were some differences in the results of the three databases used to detect resistance genes in this study. For this reason, it is important to analyze different databases and compare the database results in studies while performing whole genome sequencing. Further studies and updates using different databases will increase the compatibility between the databases.

High throughput single cell long-read sequencing analyses of same-cell genotypes and phenotypes in human tumors

Single-cell nanopore sequencing of full-length mRNAs transforms single-cell multi-omics studies. However, challenges include high sequencing errors and dependence on short-reads and/or barcode whitelists. To address these, we develop scNanoGPS to calculate same-cell genotypes (mutations) and phenotypes (gene/isoform expressions) without short-read nor whitelist guidance. We apply scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell-lines. Standalone, scNanoGPS deconvolutes error-prone long-reads into single-cells and single-molecules, and simultaneously accesses both phenotypes and genotypes of individual cells. Our analyses reveal that tumor and stroma/immune cells express distinct combination of isoforms (DCIs). In a kidney tumor, we identify 924 DCI genes involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Transcriptome-wide mutation analyses identify many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting the critical roles of different mutant populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing technologies.

Roving methyltransferases generate a mosaic epigenetic landscape and influence evolution in Bacteroides fragilis group

Three types of DNA methyl modifications have been detected in bacterial genomes, and mechanistic studies have demonstrated roles for DNA methylation in physiological functions ranging from phage defense to transcriptional control of virulence and host-pathogen interactions. Despite the ubiquity of methyltransferases and the immense variety of possible methylation patterns, epigenomic diversity remains unexplored for most bacterial species. Members of the Bacteroides fragilis group (BFG) reside in the human gastrointestinal tract as key players in symbiotic communities but also can establish anaerobic infections that are increasingly multi-drug resistant. In this work, we utilize long-read sequencing technologies to perform pangenomic (n = 383) and panepigenomic (n = 268) analysis of clinical BFG isolates cultured from infections seen at the NIH Clinical Center over four decades. Our analysis reveals that single BFG species harbor hundreds of DNA methylation motifs, with most individual motif combinations occurring uniquely in single isolates, implying immense unsampled methylation diversity within BFG epigenomes. Mining of BFG genomes identified more than 6000 methyltransferase genes, approximately 1000 of which were associated with intact prophages. Network analysis revealed substantial gene flow among disparate phage genomes, implying a role for genetic exchange between BFG phages as one of the ultimate sources driving BFG epigenome diversity.

Mge-cluster: a reference-free approach for typing bacterial plasmids.

Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However, high-resolution population-wide analysis of plasmids has only become accessible recently with the advent of scalable long-read sequencing technology. Current typing methods for the classification of plasmids remain limited in their scope which motivated us to develop a computationally efficient approach to simultaneously recognize novel types and classify plasmids into previously identified groups. Here, we introduce mge-cluster that can easily handle thousands of input sequences which are compressed using a unitig representation in a de Bruijn graph. Our approach offers a faster runtime than existing algorithms, with moderate memory usage, and enables an intuitive visualization, classification and clustering scheme that users can explore interactively within a single framework. Mge-cluster platform for plasmid analysis can be easily distributed and replicated, enabling a consistent labelling of plasmids across past, present, and future sequence collections. We underscore the advantages of our approach by analysing a population-wide plasmid data set obtained from the opportunistic pathogen Escherichia coli, studying the prevalence of the colistin resistance gene mcr-1.1 within the plasmid population, and describing an instance of resistance plasmid transmission within a hospital environment.

Complete Genome Sequencing of Three Clade-1 Xanthomonads Reveals Genetic Determinants for a Lateral Flagellin and the Biosynthesis of Coronatine-Like Molecules in Xanthomonas.

Evolutionarily, early-branching xanthomonads, also referred to as clade-1 xanthomonads, include major plant pathogens, most of which colonize monocotyledonous plants. Seven species have been validly described, among them the two sugarcane pathogens Xanthomonas albilineans and Xanthomonas sacchari, as well as Xanthomonas translucens, which infects small-grain cereals and diverse grasses but also asparagus and pistachio trees. Single-gene sequencing and genomic approaches have indicated that this clade likely contains more, yet-undescribed species. In this study, we sequenced representative strains of three novel species using long-read sequencing technology. Xanthomonas campestris pv. phormiicola strain CFBP 8444 causes bacterial streak on New Zealand flax, another monocotyledonous plant. Xanthomonas sp. strain CFBP 8443 has been isolated from common bean, and Xanthomonas sp. strain CFBP 8445 originated from banana. Complete assemblies of the chromosomes confirmed their unique phylogenetic position within clade1 of Xanthomonas. Genome mining revealed novel genetic features, hitherto undescribed in other members of the Xanthomonas genus. In strain CFBP 8444, we identified genes related to the synthesis of coronatine-like compounds, a phytotoxin produced by several pseudomonads, which raises interesting questions about the evolution and pathogenicity of this pathogen. Furthermore, strain CFBP 8444 was found to contain a second, atypical flagellar gene cluster in addition to the canonical flagellar gene cluster. Overall, this research represents an important step toward better understanding the evolutionary history and biology of early-branching xanthomonads.

JTK: targeted diploid genome assembler.

Diploid assembly, or determining sequences of homologous chromosomes separately, is essential to elucidate genetic differences between haplotypes. One approach is to call and phase single nucleotide variants (SNVs) on a reference sequence. However, this approach becomes unstable on large segmental duplications (SDs) or structural variations (SVs) because the alignments of reads deriving from these regions tend to be unreliable. Another approach is to use highly accurate PacBio HiFi reads to output diploid assembly directly. Nonetheless, HiFi reads cannot phase homozygous regions longer than their length and require oxford nanopore technology (ONT) reads or Hi-C to produce a fully phased assembly. Is a single long-read sequencing technology sufficient to create an accurate diploid assembly? Here, we present JTK, a megabase-scale diploid genome assembler. It first randomly samples kilobase-scale sequences (called 'chunks') from the long reads, phases variants found on them, and produces two haplotypes. The novel idea of JTK is to utilize chunks to capture SNVs and SVs simultaneously. From 60-fold ONT reads on the HG002 and a Japanese sample, it fully assembled two haplotypes with approximately 99.9% accuracy on the histocompatibility complex (MHC) and the leukocyte receptor complex (LRC) regions, which was impossible by the reference-based approach. In addition, in the LRC region on a Japanese sample, JTK output an assembly of better contiguity than those built from high-coverage HiFi+Hi-C. In the coming age of pan-genomics, JTK would complement the reference-based phasing method to assemble the difficult-to-assemble but medically important regions. JTK is available at https://github.com/ban-m/jtk, and the datasets are available at https://doi.org/10.5281/zenodo.7790310 or JGAS000580 in DDBJ.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Using nanopore sequencing to identify fungi from clinical samples with high phylogenetic resolution

The study of microbiota has been revolutionized by the development of DNA metabarcoding. This sequence-based approach enables the direct detection of microorganisms without the need for culture and isolation, which significantly reduces analysis time and offers more comprehensive taxonomic profiles across broad phylogenetic lineages. While there has been an accumulating number of researches on bacteria, molecular phylogenetic analysis of fungi still remains challenging due to the lack of standardized tools and the incompleteness of reference databases limiting the accurate and precise identification of fungal taxa. Here, we present a DNA metabarcoding workflow for characterizing fungal microbiota with high taxonomic resolution. This method involves amplifying longer stretches of ribosomal RNA operons and sequencing them using nanopore long-read sequencing technology. The resulting reads were error-polished to generate consensus sequences with 99.5–100% accuracy, which were then aligned against reference genome assemblies. The efficacy of this method was explored using a polymicrobial mock community and patient-derived specimens, demonstrating the marked potential of long-read sequencing combined with consensus calling for accurate taxonomic classification. Our approach offers a powerful tool for the rapid identification of pathogenic fungi and has the promise to significantly improve our understanding of the role of fungi in health and disease.

Molecular characterization of a novel 83.9-kb deletion of the α-globin upstream regulatory elements by long-read sequencing

Inherited deletions of upstream regulatory elements of α-globin genes give rise to α-thalassemia, which is an autosomal recessive monogenic disease. However, conventional thalassemia target diagnosis often fails to identify these rare deletions. Here we reported a family with two previous pregnancies of Hb Bart's hydrops fetalis and was seeking for prenatal diagnosis during the third pregnancy. Both parents had low level of Hemoglobin A2 indicating α-thalassemia. Conventional Gap-PCR and PCR-reverse dot blot showed the father carried –SEA deletion but did not identify any variants in the mother. Multiplex ligation-dependent probe amplification identified a deletion containing two HS-40 probes but could not determine the exact region. Finally, a long-read sequencing (LRS)-based approach directly identified that the exact deletion region was chr16: 48,642-132,584, which was located in the α-globin upstream regulatory elements and named (αα)JM after the Jiangmen city. Gap-PCR and Sanger sequencing confirmed the breakpoint. Both the mother and fetus from the third pregnancy carried heterozygous (αα)JM, and the fetus was normally delivered at gestational age of 39 weeks. This study demonstrated that LRS technology had great advantages over conventional target diagnosis methods for identifying rare thalassemia variants and assisted better carrier screening and prenatal diagnosis of thalassemia.

Y chromosome sequence and epigenomic reconstruction across human populations

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for seven major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications among the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets.

High-Quality Genome Resource of Fusarium pseudograminearum Isolate Fp22-2 by Oxford Nanopore Long-Read Sequencing.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, results in severe yield and quality losses of cereal crops in many arid and semiarid areas of the world. Limited information about the genome of F. pseudograminearum restricts the pathogenesis research and breeding of disease-resistant wheat varieties. In this study, a high-quality genome assembly of F. pseudograminearum isolate Fp22-2 was generated using Oxford Nanopore long-read sequencing technology. The assembled nuclear genome of Fp22-2 is 37.33 Mb with a repeat content of 3.69% and is divided into four contigs with a k-mer completeness score of 97.2% and a base quality accuracy of >99.99%. A total of 14,475 protein-coding genes (BUSCO completeness score, 99.9%) were predicted and functionally annotated. Moreover, genes encoding pathogenic proteins, including effector proteins and carbohydrate-active enzymes, and secondary metabolic gene clusters were identified. Overall, the high-quality genome assembly and gene annotation provided here will allow further investigation of the biology of F. pseudograminearum and lead to the development of new control options for FCR.