Co-transcriptional splicing regulates 3' end cleavage during mammalian erythropoiesis.

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3' end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improved both splicing and 3' end cleavage of individual ß-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.

The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

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.

Beyond the exome: What's next in diagnostic testing for Mendelian conditions.

Despite advances in clinical genetic testing, including the introduction of exome sequencing (ES), more than 50% of individuals with a suspected Mendelian condition lack a precise molecular diagnosis. Clinical evaluation is increasingly undertaken by specialists outside of clinical genetics, often occurring in a tiered fashion and typically ending after ES. The current diagnostic rate reflects multiple factors, including technical limitations, incomplete understanding of variant pathogenicity, missing genotype-phenotype associations, complex gene-environment interactions, and reporting differences between clinical labs. Maintaining a clear understanding of the rapidly evolving landscape of diagnostic tests beyond ES, and their limitations, presents a challenge for non-genetics professionals. Newer tests, such as short-read genome or RNA sequencing, can be challenging to order, and emerging technologies, such as optical genome mapping and long-read DNA sequencing, are not available clinically. Furthermore, there is no clear guidance on the next best steps after inconclusive evaluation. Here, we review why a clinical genetic evaluation may be negative, discuss questions to be asked in this setting, and provide a framework for further investigation, including the advantages and disadvantages of new approaches that are nascent in the clinical sphere. We present a guide for the next best steps after inconclusive molecular testing based upon phenotype and prior evaluation, including when to consider referral to research consortia focused on elucidating the underlying cause of rare unsolved genetic disorders.

Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing.

Spinal muscular atrophy, a leading cause of early infant death, is caused by bi-allelic mutations of SMN1. Sequence analysis of SMN1 is challenging due to high sequence similarity with its paralog SMN2. Both genes have variable copy numbers across populations. Furthermore, without pedigree information, it is currently not possible to identify silent carriers (2+0) with two copies of SMN1 on one chromosome and zero copies on the other. We developed Paraphase, an informatics method that identifies full-length SMN1 and SMN2 haplotypes, determines the gene copy numbers, and calls phased variants using long-read PacBio HiFi data. The SMN1 and SMN2 copy-number calls by Paraphase are highly concordant with orthogonal methods (99.2% for SMN1 and 100% for SMN2). We applied Paraphase to 438 samples across 5 ethnic populations to conduct a population-wide haplotype analysis of these highly homologous genes. We identified major SMN1 and SMN2 haplogroups and characterized their co-segregation through pedigree-based analyses. We identified two SMN1 haplotypes that form a common two-copy SMN1 allele in African populations. Testing positive for these two haplotypes in an individual with two copies of SMN1 gives a silent carrier risk of 88.5%, which is significantly higher than the currently used marker (1.7%-3.0%). Extending beyond simple copy-number testing, Paraphase can detect pathogenic variants and enable potential haplotype-based screening of silent carriers through statistical phasing of haplotypes into alleles. Future analysis of larger population data will allow identification of more diverse haplotypes and genetic markers for silent carriers.

A survey on computational strategies for genome-resolved gut metagenomics.

Recovering high-quality metagenome-assembled genomes (HQ-MAGs) is critical for exploring microbial compositions and microbe-phenotype associations. However, multiple sequencing platforms and computational tools for this purpose may confuse researchers and thus call for extensive evaluation. Here, we systematically evaluated a total of 40 combinations of popular computational tools and sequencing platforms (i.e. strategies), involving eight assemblers, eight metagenomic binners and four sequencing technologies, including short-, long-read and metaHiC sequencing. We identified the best tools for the individual tasks (e.g. the assembly and binning) and combinations (e.g. generating more HQ-MAGs) depending on the availability of the sequencing data. We found that the combination of the hybrid assemblies and metaHiC-based binning performed best, followed by the hybrid and long-read assemblies. More importantly, both long-read and metaHiC sequencings link more mobile elements and antibiotic resistance genes to bacterial hosts and improve the quality of public human gut reference genomes with 32% (34/105) HQ-MAGs that were either of better quality than those in the Unified Human Gastrointestinal Genome catalog version 2 or novel.

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.

Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples.

BACKGROUND: Sequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively. RESULTS: With both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing. CONCLUSIONS: The results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Physiological responses to drought stress of three pine species and comparative transcriptome analysis of Pinus yunnanensis var. pygmaea.

Drought stress can significantly affect plant growth, development, and yield. Fewer comparative studies have been conducted between different species of pines, particularly involving Pinus yunnanensis var. pygmaea (P. pygmaea). In this study, the physiological indices, photosynthetic pigment and related antioxidant enzyme changes in needles from P. pygmaea, P. elliottii and P. massoniana under drought at 0, 7, 14, 21, 28 and 35 d, as well as 7 days after rehydration, were measured. The PacBio single-molecule real-time (SMRT) and Illumina RNA sequencing were used to uncover the gene expression differences in P. pygmaea under drought and rehydration conditions. The results showed that the total antioxidant capacity (TAOC) of P. pygmaea was significantly higher than P. massoniana and P. elliottii. TAOC showed a continuous increase trend across all species. Soluble sugar (SS), starch content and non-structural carbohydrate (NSC) of all three pines displayed a "W" pattern, declining initially, increasing, and then decreasing again. P. pygmaea exhibits stronger drought tolerance and greater recovery ability under prolonged drought conditions. Through the PacBio SMRT-seq, a total of 50,979 high-quality transcripts were generated, and 6,521 SSR and 5,561 long non-coding RNAs (LncRNAs) were identified. A total of 2310, 1849, 5271, 5947, 7710, and 6854 differentially expressed genes (DEGs) were identified compared to the control (Pp0D) in six pair-wise comparisons of treatment versus control. bHLH, NAC, ERF, MYB_related, C3H transcription factors (TFs) play an important role in drought tolerance of P. pygmaea. KEGG enrichment analysis and Gene set enrichment analysis (GSEA) analysis showed that P. pygmaea may respond to drought by enhancing metabolic processes such as ABA signaling pathway, alpha-linolenic acid. Weighted gene co-expression network analysis (WGCNA) revealed GST, CAT, LEC14B, SEC23 were associated with antioxidant enzyme activity and TAOC. This study provides a basis for further research on drought tolerance differences among coniferous species.

De novo assembly and annotation of the Patagonian toothfish (Dissostichus eleginoides) genome.

BACKGROUND: Patagonian toothfish (Dissostichus eleginoides) is an economically and ecologically important fish species in the family Nototheniidae. Juveniles occupy progressively deeper waters as they mature and grow, and adults have been caught as deep as 2500 m, living on or in just above the southern shelves and slopes around the sub-Antarctic islands of the Southern Ocean. As apex predators, they are a key part of the food web, feeding on a variety of prey, including krill, squid, and other fish. Despite its importance, genomic sequence data, which could be used for more accurate dating of the divergence between Patagonian and Antarctic toothfish, or establish whether it shares adaptations to temperature with fish living in more polar or equatorial climes, has so far been limited. RESULTS: A high-quality D. eleginoides genome was generated using a combination of Illumina, PacBio and Omni-C sequencing technologies. To aid the genome annotation, the transcriptome derived from a variety of toothfish tissues was also generated using both short and long read sequencing methods. The final genome assembly was 797.8 Mb with a N50 scaffold length of 3.5 Mb. Approximately 31.7% of the genome consisted of repetitive elements. A total of 35,543 putative protein-coding regions were identified, of which 50% have been functionally annotated. Transcriptomics analysis showed that approximately 64% of the predicted genes (22,617 genes) were found to be expressed in the tissues sampled. Comparative genomics analysis revealed that the anti-freeze glycoprotein (AFGP) locus of D. eleginoides does not contain any AFGP proteins compared to the same locus in the Antarctic toothfish (Dissostichus mawsoni). This is in agreement with previously published results looking at hybridization signals and confirms that Patagonian toothfish do not possess AFGP coding sequences in their genome. CONCLUSIONS: We have assembled and annotated the Patagonian toothfish genome, which will provide a valuable genetic resource for ecological and evolutionary studies on this and other closely related species.

Comparative transcriptomic analysis delineates adaptation strategies of Rana kukunoris toward cold stress on the Qinghai-Tibet Plateau.

BACKGROUND: Cold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter. RESULTS: Transcriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring. CONCLUSIONS: Based on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs.

Identification of key genes for triacylglycerol biosynthesis and storage in herbaceous peony (Paeonia lactifolra Pall.) seeds based on full-length transcriptome.

BACKGROUND: The herbaceous peony (Paeonia lactiflora Pall.) is extensively cultivated in China due to its root being used as a traditional Chinese medicine known as 'Radix Paeoniae Alba'. In recent years, it has been discovered that its seeds incorporate abundant unsaturated fatty acids, thereby presenting a potential new oilseed plant. Surprisingly, little is known about the full-length transcriptome sequencing of Paeonia lactiflora, limiting research into its gene function and molecular mechanisms. RESULTS: A total of 484,931 Reads of Inserts (ROI) sequences and 1,455,771 full-Length non-chimeric reads (FLNC) sequences were obtained for CDS prediction, TF analysis, SSR analysis and lncRNA identification. In addition, gene function annotation and gene structure analysis were performed. A total of 4905 transcripts were related to lipid metabolism biosynthesis pathway, belonging to 28 enzymes. We use these data to identify 10 oleosin (OLE) and 5 diacylglycerol acyltransferase (DGAT) gene members after de-redundancy. The analysis of physicochemical properties and secondary structure showed them similarity in gene family respectively. The phylogenetic analysis showed that the distribution of OLE and DGAT family members was roughly the same as that of Arabidopsis. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed expression changes in different seed development stages, and showed a trend of increasing and then decreasing. CONCLUSION: In summary, these results provide new insights into the molecular mechanism of triacylglycerol (TAG) biosynthesis and storage during the seedling stage in Paeonia lactiflora. It provides theoretical references for selecting and breeding oil varieties and understanding the functions of oil storage as well as lipid synthesis related genes in Paeonia lactiflora.

Copy number variation and elevated genetic diversity at immune trait loci in Atlantic and Pacific herring.

BACKGROUND: Genome-wide comparisons of populations are widely used to explore the patterns of nucleotide diversity and sequence divergence to provide knowledge on how natural selection and genetic drift affect the genome. In this study we have compared whole-genome sequencing data from Atlantic and Pacific herring, two sister species that diverged about 2 million years ago, to explore the pattern of genetic differentiation between the two species. RESULTS: The genome comparison of the two species revealed high genome-wide differentiation but with islands of remarkably low genetic differentiation, as measured by an FST analysis. However, the low FST observed in these islands is not caused by low interspecies sequence divergence (dxy) but rather by exceptionally high estimated intraspecies nucleotide diversity (π). These regions of low differentiation and elevated nucleotide diversity, termed high-diversity regions in this study, are not enriched for repeats but are highly enriched for immune-related genes. This enrichment includes genes from both the adaptive immune system, such as immunoglobulin, T-cell receptor and major histocompatibility complex genes, as well as a substantial number of genes with a role in the innate immune system, e.g. novel immune-type receptor, tripartite motif and tumor necrosis factor receptor genes. Analysis of long-read based assemblies from two Atlantic herring individuals revealed extensive copy number variation in these genomic regions, indicating that the elevated intraspecies nucleotide diversities were partially due to the cross-mapping of short reads. CONCLUSIONS: This study demonstrates that copy number variation is a characteristic feature of immune trait loci in herring. Another important implication is that these loci are blind spots in classical genome-wide screens for genetic differentiation using short-read data, not only in herring, likely also in other species harboring qualitatively similar variation at immune trait loci. These loci stood out in this study because of the relatively high genome-wide baseline for FST values between Atlantic and Pacific herring.

Annotated genome and transcriptome of the endangered Caribbean mountainous star coral (Orbicella faveolata) using PacBio long-read sequencing.

Long-read sequencing is revolutionizing de-novo genome assemblies, with continued advancements making it more readily available for previously understudied, non-model organisms. Stony corals are one such example, with long-read de-novo genome assemblies now starting to be publicly available, opening the door for a wide array of 'omics-based research. Here we present a new de-novo genome assembly for the endangered Caribbean star coral, Orbicella faveolata, using PacBio circular consensus reads. Our genome assembly improved the contiguity (51 versus 1,933 contigs) and complete and single copy BUSCO orthologs (93.6% versus 85.3%, database metazoa_odb10), compared to the currently available reference genome generated using short-read methodologies. Our new de-novo assembled genome also showed comparable quality metrics to other coral long-read genomes. Telomeric repeat analysis identified putative chromosomes in our scaffolded assembly, with these repeats at either one, or both ends, of scaffolded contigs. We identified 32,172 protein coding genes in our assembly through use of long-read RNA sequencing (ISO-seq) of additional O. faveolata fragments exposed to a range of abiotic and biotic treatments, and publicly available short-read RNA-seq data. With anthropogenic influences heavily affecting O. faveolata, as well as its increasing incorporation into reef restoration activities, this updated genome resource can be used for population genomics and other 'omics analyses to aid in the conservation of this species.

Single-molecule Sequencing of an Animal Mitochondrial Genome Reveals Chloroplast-like Architecture and Repeat-mediated Recombination.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus. These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa-SSC-IRb structure revealed the occurrence of a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution.

Y-Linked Copy Number Polymorphism of Target of Rapamycin Is Associated with Sexual Size Dimorphism in Seed Beetles.

The Y chromosome is theorized to facilitate evolution of sexual dimorphism by accumulating sexually antagonistic loci, but empirical support is scarce. Due to the lack of recombination, Y chromosomes are prone to degenerative processes, which poses a constraint on their adaptive potential. Yet, in the seed beetle, Callosobruchus maculatus segregating Y linked variation affects male body size and thereby sexual size dimorphism (SSD). Here, we assemble C. maculatus sex chromosome sequences and identify molecular differences associated with Y-linked SSD variation. The assembled Y chromosome is largely euchromatic and contains over 400 genes, many of which are ampliconic with a mixed autosomal and X chromosome ancestry. Functional annotation suggests that the Y chromosome plays important roles in males beyond primary reproductive functions. Crucially, we find that, besides an autosomal copy of the gene target of rapamycin (TOR), males carry an additional TOR copy on the Y chromosome. TOR is a conserved regulator of growth across taxa, and our results suggest that a Y-linked TOR provides a male specific opportunity to alter body size. A comparison of Y haplotypes associated with male size difference uncovers a copy number variation for TOR, where the haplotype associated with decreased male size, and thereby increased sexual dimorphism, has two additional TOR copies. This suggests that sexual conflict over growth has been mitigated by autosome to Y translocation of TOR followed by gene duplications. Our results reveal that despite of suppressed recombination, the Y chromosome can harbor adaptive potential as a male-limited supergene.

Chromosome-scale Genome Assembly and Characterization of Top-quality Japanese Green Tea Cultivar 'Seimei'.

Japanese green tea, an essential beverage in Japanese culture, is characterized by the initial steaming of freshly harvested leaves during production. This process efficiently inactivates endogenous enzymes such as polyphenol oxidases, resulting in the production of sencha, gyokuro, and matcha that preserves the vibrant green color of young leaves. Although genome sequences of several tea cultivars and germplasms have been published, no reference genome sequences are available for Japanese green tea cultivars. Here, we constructed a reference genome sequence of the cultivar 'Seimei', which is used to produce high-quality Japanese green tea. Using the PacBio HiFi and Hi-C technologies for chromosome-scale genome assembly, we obtained 15 chromosome sequences with a total genome size of 3.1 Gb and an N50 of 214.9 Mb. By analyzing the genomic diversity of 23 Japanese tea cultivars and lines, including the leading green tea cultivars 'Yabukita' and 'Saemidori', revealed several candidate genes that could be related to the characteristics of Japanese green tea. The reference genome of 'Seimei' and information on genomic diversity of Japanese green tea cultivars should provide crucial information for effective breeding of such cultivars in the future.

Diversity of the Rysto gene conferring resistance to potato virus Y in wild relatives of potato.

BACKGROUND: Potato virus Y (PVY) is among the economically most damaging viral pathogen in production of potato (Solanum tuberosum) worldwide. The gene Rysto derived from the wild potato relative Solanum stoloniferum confers extreme resistance to PVY. RESULTS: The presence and diversity of Rysto were investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species) using PacBio amplicon sequencing. A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of 10 Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363). The 55 Rysto-like sequences showed 89.87 to 99.98% nucleotide identity to the Rysto reference gene, and these encoded in total 45 unique protein sequences. While Rysto-like26 identified in Alicja, Bzura, White Lady and Rysto-like16 in PW363 encode a protein identical to the Rysto reference, the remaining 44 predicted Rysto-like proteins were 65.93 to 99.92% identical to the reference. Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in the resistant control cultivars. The TIR and NB-ARC domains were the most conserved within the Rysto-like proteins, while the LRR and C-JID domains were more variable. Several Solanum species, including S. antipoviczii and S. hougasii, showed resistance to PVY. This study demonstrated Hyoscyamus niger, a Solanaceae species distantly related to Solanum, as a host of PVY. CONCLUSIONS: The new Rysto-like variants and the identified PVY resistant potato genotypes are potential resistance sources against PVY in potato breeding. Identification of H. niger as a host for PVY is important for cultivation of this plant, studies on the PVY management, its ecology, and migrations. The amplicon sequencing based on PacBio SMRT and the following data analysis pipeline described in our work may be applied to obtain the nucleotide sequences and analyze any full-length genes from any, even polyploid, organisms.

Unraveling metagenomics through long-read sequencing: a comprehensive review.

The study of microbial communities has undergone significant advancements, starting from the initial use of 16S rRNA sequencing to the adoption of shotgun metagenomics. However, a new era has emerged with the advent of long-read sequencing (LRS), which offers substantial improvements over its predecessor, short-read sequencing (SRS). LRS produces reads that are several kilobases long, enabling researchers to obtain more complete and contiguous genomic information, characterize structural variations, and study epigenetic modifications. The current leaders in LRS technologies are Pacific Biotechnologies (PacBio) and Oxford Nanopore Technologies (ONT), each offering a distinct set of advantages. This review covers the workflow of long-read metagenomics sequencing, including sample preparation (sample collection, sample extraction, and library preparation), sequencing, processing (quality control, assembly, and binning), and analysis (taxonomic annotation and functional annotation). Each section provides a concise outline of the key concept of the methodology, presenting the original concept as well as how it is challenged or modified in the context of LRS. Additionally, the section introduces a range of tools that are compatible with LRS and can be utilized to execute the LRS process. This review aims to present the workflow of metagenomics, highlight the transformative impact of LRS, and provide researchers with a selection of tools suitable for this task.

A chromosome-level genome assembly for the smoky rubyspot damselfly (Hetaerina titia).

Smoky rubyspot damselflies (Hetaerina titia Drury, 1773) are one of the most commonly encountered odonates along streams and rivers on both slopes of Central America and the Atlantic drainages in the United States and southern Canada. Owing to their highly variable wing pigmentation, they have become a model system for studying sexual selection and interspecific behavioral interference. Here, we sequence and assemble the genome of a female smoky rubyspot. Of the primary assembly (i.e. the principle pseudohaplotype), 98.8% is made up of 12 chromosomal pseudomolecules (2N = 22A + X). There are 75 scaffolds in total, an N50 of 120 Mb, a contig-N50 of 0.64 Mb, and a high arthropod BUSCO score [C: 97.6% (S: 97.3%, D: 0.3%), F: 0.8%, M: 1.6%]. We then compare our assembly to that of the blue-tailed damselfly genome (Ischnura elegans), the most complete damselfly assembly to date, and a recently published assembly for an American rubyspot damselfly (Hetaerina americana). Collectively, these resources make Hetaerina a genome-enabled genus for further studies of the ecological and evolutionary forces shaping biological diversity.