Long-read Sequencing Research Articles

The Utility of Long-Read Sequencing in Diagnosing Early Onset Parkinson's Disease.

Variants in PRKN and PINK1 are the leading cause of early-onset autosomal recessive Parkinson's disease, yet many cases remain genetically unresolved. We previously identified a 7 megabases complex structural variant in a pair of monozygotic twins using Oxford Nanopore Technologies (ONT) long-read sequencing. This study aims to determine if ONT long-read sequencing can detect a second variant in other unresolved early-onset Parkinson's disease (EOPD) cases with 1 heterozygous PRKN or PINK1 variant. ONT long-read sequencing was performed on EOPD patients with 1 reported PRKN/PINK1 pathogenic variant, with onset age under 50. Positive controls included EOPD patients with 2 known PRKN pathogenic variants. Initial testing involved short-read targeted panel sequencing for single nucleotide variants and multiplex ligation-dependent probe amplification for copy number variants. A total of 47 patients were studied (PRKN "one-variant," n = 23; PINK1 "one-variant," n = 12; PRKN "two-variants," n = 12). ONT long-read sequencing identified a second pathogenic variant in 26% of PRKN "one-variant" patients (6/23), but none in PINK1 "one-variant" patients (0/12). Detected variants included 1 complex inversion, 2 structural variant overlaps, and 3 duplications. In the PRKN "two-variants" group, both variants were identified in all patients (100%, 12/12). ONT long-read sequencing effectively identifies pathogenic structural variants in the PRKN locus missed by conventional methods. It should be considered for unresolved EOPD cases when a second variant is not detected through conventional approaches. ANN NEUROL 2024.

Long-read genome sequencing resolves complex genomic rearrangements in rare genetic syndromes

Long-read sequencing can often overcome the deficiencies in routine microarray or short-read technologies in detecting complex genomic rearrangements. Here we used Pacific Biosciences circular consensus sequencing to resolve complex rearrangements in two patients with rare genetic anomalies. Copy number variants (CNVs) identified by clinical microarray —chr8p deletion and chr8q duplication in patient 1, and interstitial deletions of chr18q in patient 2—were suggestive of underlying rearrangements. Long-read genome sequencing not only confirmed these CNVs but also revealed their genomic structures. In patient 1, we resolved a novel recombinant chromosome 8 (Rec8)-like rearrangement with a 3.43 Mb chr8q terminal duplication that was linked to a 7.25–8.21 Mb chr8p terminal deletion. In patient 2, we uncovered a novel complex rearrangement involving a 1.17 Mb rearranged segment and four interstitial deletions ranging from 9 bp to 12.39 Mb. Our results underscore the diversity of clinically relevant structural rearrangements and the power of long-read sequencing in unraveling their nuanced architectures.

Taurine pangenome uncovers a segmental duplication upstream of KIT associated with depigmentation in white-headed cattle.

Cattle have been selectively bred for coat color, spotting, and depigmentation patterns. The assumed autosomal dominant inherited genetic variants underlying the characteristic white head of Fleckvieh, Simmental, and Hereford cattle have not been identified yet, although the contribution of structural variation upstream the KIT gene has been proposed. Here, we construct a graph pangenome from 24 haplotype assemblies representing seven taurine cattle breeds to identify and characterize the white head-associated locus for the first time based on long-read sequencing data and pangenome analyses. We introduce a pangenome-wide association mapping approach which examines assembly path similarities within the graph to reveal an association between two most likely serial alleles of a complex structural variant 66 kb upstream KIT and facial depigmentation. The complex structural variant contains a variable number of tandemly duplicated 14.3 kb repeats, consisting of LTRs, LINEs, and other repetitive elements, leading to misleading alignments of short and long reads when using a linear reference. We align 250 short-read sequencing samples spanning 15 cattle breeds to the pangenome graph, further validating that the alleles of the structural variant segregate with head depigmentation. We estimate an increased count of repeats in Hereford relative to Simmental and other white-headed cattle breeds from the graph alignment coverage, suggesting a large under-assembly in the current Hereford-based cattle reference genome which had fewer copies. Our work shows that exploiting assembly path similarities within graph pangenomes can reveal trait-associated complex structural variants.

De novo assembly of a near-complete genome of aquatic vegetable Zizania latifolia in the Yangtze River Basin

The cultivated Zizania latifolia, an aquatic vegetable prevalent in the Yangtze River Basin, represents a unique plant-fungus complex whose domestication is associated with host-parasite co-evolution. In this study, we present a high-quality, chromosome-scale genome assembly of cultivated Z. latifolia. We employed PacBio long-read sequencing and Hi-C technology to generate ~578.42 Mb genome assembly, which contains 47.59% repeat sequences with a contig N50 of ~33.75 Mb. The contigs were successfully clustered into 17 chromosomal-sized scaffolds with a GC content of 43.26%, showing 98.39% completeness in BUSCO analysis. In total, we predicted 39,934 protein-coding genes, 88.79% of which could be functionally annotated. This genome assembly provides a valuable resource for unraveling Z. latifolia’s domestication process, and advances our understanding of the evolutionary history and agricultural potential of Z. latifolia.

CNVizard—a lightweight streamlit application for an interactive analysis of copy number variants

BackgroundMethods to call, analyze and visualize copy number variations (CNVs) from massive parallel sequencing data have been widely adopted in clinical practice and genetic research. To enable a streamlined analysis of CNV data, comprehensive annotations and good visualizations are indispensable. The ability to detect single exon CNVs is another important feature for genetic testing. Nonetheless, most available open-source tools come with limitations in at least one of these areas. One additional drawback is that available tools deliver data in an unstructured and static format which requires subsequent visualization and formatting efforts.ResultsHere we present CNVizard, an interactive Streamlit app allowing a comprehensive visualization of CNVkit data. Furthermore, combining CNVizard with the CNVand pipeline allows the annotation and visualization of CNV or SV VCF files from any CNV caller.ConclusionCNVizard, in combination with CNVand, enables the comprehensive and streamlined analysis of short- and long-read sequencing data and provide an intuitive webapp-like experience enabling an interactive visualization of CNV data.

Uncovering the complexity of structural variants in four individuals with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is an increasingly recognized childhood developmental disorder. Despite extensive study, causal variants and molecular diagnosis remain elusive. There is both heterogeneity of the phenotype, as well as the genetic landscape associated with phenotype, which includes both inherited and de-novo mutations. Currently, diagnosis is complex and behaviourally based, oftentimes occurring years after the ideal 1-2 years age. Structural variants (SVs) are large and sometimes complex genomic variants that are likely underrepresented contributors to ASD due to the limitations of short-read DNA sequencing, such as alignment in repetitive regions and regions with GC bias. Here, we performed long read sequencing (LRS) on four individuals with autism spectrum disorder to delineate SV complexity and determine precise breakpoints for SVs, which was not possible with short read sequencing (SRS). We use LRS to interrogate the methylation pattern associated with the SVs and phase the SV haplotypes to further clarify their contribution to disorder. LRS allows insight into the genome and methylome that allow us to uncover variant complexity and contribution that was previously unseen with SRS. Ultimately, this furthers precision diagnosis and contributes to individualized treatment for affected individuals and their families within the clinic.

RNA isoform diversity in human neurodegenerative diseases.

Single-nucleus RNA-sequencing (snRNA-seq) has revealed new levels of cellular organization and diversity within the human brain. However, full-length mRNA isoforms are not resolved in typical snRNA-seq analyses using short-read sequencing that cannot capture full-length transcripts. Here we combine standard 10X Genomics short-read snRNA-seq with targeted PacBio long-read snRNA-seq to examine isoforms of genes associated with neurological diseases at the single-cell level from prefrontal cortex samples of diseased and non-diseased human brain, assessing over 165,000 cells. Samples from 25 post-mortem donors with Alzheimer's disease (AD), dementia with Lewy bodies (DLB), or Parkinson's disease (PD), along with age-matched controls were compared. Analysis of the short-read libraries identified shared and distinct gene expression changes across the diseases. The same libraries were then assayed using enrichment probes to target 50 disease-related genes followed by long-read PacBio sequencing, enabling linkage between cell type and isoform expression. Vast mRNA isoform diversity was observed in all 50 targeted genes, even those that were not differentially expressed in the short-read data. We also developed an informatics method for detection of isoform structural differences in novel isoforms vs. the reference annotation. These data expand available single-cell datasets of the human prefrontal cortical transcriptome with combined short- and long-read sequencing across AD, DLB, and PD, revealing increased mRNA isoform diversity that may contribute to disease features and could potentially represent therapeutic targets for neurodegenerative diseases.Significance Statement Limited comparisons using single-cell transcriptomics analysis have been conducted amongst common neurodegenerative diseases. Here we identify new cell type and disease relationships involving known and novel mRNA isoforms by profiling single nuclei from human prefrontal cortices of Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies and non-diseased controls. Cell-type-specific RNA isoform diversity across the different diseases was examined using the combination of short-read snRNA-seq and targeted long-read single-nucleus isoform sequencing. We identified myriad novel transcripts that highlight an untapped understanding of RNA isoform diversity that exists within the brain and potentially contribute to human neurodegenerative diseases.

Detection and characterization of the types of CYP21A1P/CYP21A2 and TNXA/TNXB fused genes by long-read sequencing among children with Steroid 21-hydroxylase deficiency

To assess the diagnostic efficiency of long-read sequencing (LRS) for the determination of CYP21A1P/CYP21A2 and TNXA/TNXB fusion genotypes among children with 21-hydroxylase deficiency (21-OHD) and explore their clinical characteristics. LRS sequencing was carried out on 30 children diagnosed with 21-OHD at the Department of Endocrinology, Fujian Children's Hospital between November 2022 and September 2023 by clinical symptoms or conventional Sanger sequencing combined with multiple ligation-dependent probe amplification (MLPA). The results of the two methods were compared. Clinical data of the children were collected and analyzed. This study has been approved by the Medical Ethics Committee of the Fujian Children's Hospital (Ethic No. 2022ETKLR10024). Of the 30 children with 21-OHD, 11 (36.7%) were found to carry CYP21A1P/CYP21A2 and TNXA/TNXB fusion genes by LRS. The most common type of fused CYP21A1P/CYP21A2 gene was CH-1 (72.7%), and 1 (3.3%) was found to harbor TNXA/TNXB CH-1. Eleven cases (36.7%) were found to carry large deletions by Sanger sequencing combined with MLPA, with the most common one being CYP21A2 exons 1-3 del (72.7%), which was followed by CYP21A2 exons 1-7 del (18.2%). Follow up of 11 patients carrying a fusion gene revealed that 6 were sale wasting (SW) types, 5 were simple virilizing (SV) types, whilst no non-classical (NC) type was found. Four girls had presented with central precocious puberty (CPP). One child carrying TNXA/TNXB CH-1 had presented with CAH-X syndrome. Compared with Sanger sequencing combined with MLPA detection method, LRS sequencing was able to differentiate the subtypes of CYP21A1P/CYP21A2 and TNXA/TNXB fusion genes, pinpoint the breakpoints of the deletions, and directly determine the cis-trans position without the need to analyze the genotype of the pedigree members, which has provided a reliable method for the typing of 21-OHD. As some fusion genes may retain 21-hydroxylase activity, female carriers may have a higher incidence of CPP.

Tandem repeat disorders: from diagnosis to emerging therapeutic strategies.

Tandem repeat disorders (TRDs) are genetic conditions characterized by the abnormal expansion of repetitive DNA sequences within specific genes. The growing number of identified TRDs highlights their complexity, with varied molecular mechanisms ranging from toxic protein production and repeat-associated non-AUG translation to RNA toxicity and epigenetic modifications. TRDs also exhibit unique clinical features such as reduced penetrance, anticipation, and repeat motif changes. Advances in molecular diagnostics such as long-read sequencing have significantly improved the detection of TRDs, especially for large or complex repeat expansions. Additionally, emerging therapeutic strategies, particularly antisense oligonucleotides (ASOs) and gene editing technologies, are showing great promise. ASOs in particular have demonstrated success through mechanisms like allele-specific knockdown and splice modulation. In this review, we explore the classification of TRDs, advances in diagnostics, molecular mechanisms, clinical features, and innovative therapeutic strategies, highlighting the need for further research to refine treatments and improve outcomes.

Closing the genome of Teredinibacter turnerae T7902 by long-read nanopore sequencing.

We present the complete closed circular genome sequence derived from the Oxford Nanopore sequencing of the shipworm endosymbiont, Teredinibacter turnerae T7902 (DSM 15152, ATCC 39867), originally isolated from the shipworm, Lyrodus pedicellatus (1). This sequence will aid in the comparative genomics of shipworm endosymbionts and the understanding of the host-symbiont evolution.

Naturally acquired IgG responses to Plasmodium falciparum do not target the conserved termini of the malaria vaccine candidate Merozoite Surface Protein 2

IntroductionMalaria remains a significant burden, and a fully protective vaccine against Plasmodium falciparum is critical for reducing morbidity and mortality. Antibody responses against the blood-stage antigen Merozoite Surface Protein 2 (MSP2) are associated with protection from P. falciparum malaria, but its extensive polymorphism is a barrier to its development as a vaccine candidate. New tools, such as long-read sequencing and accurate protein structure modelling allow us to study the genetic diversity and immune responses towards antigens from clinical isolates with unprecedented detail. This study sought to better understand naturally acquired MSP2-specific antibody responses.MethodsIgG responses against recombinantly expressed full-length, central polymorphic regions, and peptides derived from the conserved termini of MSP2 variants sequenced from patient isolates, were tested in plasma from travelers with recent, acute malaria and from individuals living in an endemic area of Tanzania.ResultsIgG responses towards full MSP2 and truncated MSP2 antigens were variant specific. IgG antibodies in the plasma of first-time infected or previously exposed travelers did not recognize the conserved termini of expressed MSP2 variants by ELISA, but they bound 13-amino acid long linear epitopes from the termini in a custom-made peptide array. Alphafold3 modelling suggests extensive structural heterogeneity in the conserved termini upon antigen oligomerization. IgG from individuals living in an endemic region, many who were asymptomatically infected, did not recognize the conserved termini by ELISA.DiscussionOur results suggest that responses to the variable regions are critical for the development of naturally acquired immunity towards MSP2.

Multi-omics analysis detail a submicroscopic inv(15)(q14q15) generating fusion transcripts and MEIS2 and NUSAP1 haploinsufficiency

Inversions are balanced structural variants that often remain undetected in genetic diagnostics. We present a female proband with a de novo Chromosome 15 paracentric inversion, disrupting MEIS2 and NUSAP1. The inversion was detected by short-read genome sequencing and confirmed with adaptive long-read sequencing. The breakpoint junction analysis revealed a 96 bp (bp) deletion and an 18 bp insertion in the two junctions, suggesting that the rearrangement arose through a replicative error. Transcriptome sequencing of cultured fibroblasts revealed normal MEIS2 levels and 0.61-fold decreased expression of NUSAP1. Furthermore, three fusion transcripts were detected and confirmed by Sanger sequencing. Heterozygous loss of MEIS2 (MIM# 600987) is associated with a cleft palate, heart malformations, and intellectual impairment, which overlap with the clinical symptoms observed in the proband. The observed fusion transcripts are likely non-functional, and MEIS2 haploinsufficiency is the likely disease causative mechanism. Altogether, this study’s findings illustrate the importance of including inversions in rare disease diagnostic testing and highlight the value of long read sequencing for the validation and characterization of such variants.

The ZFHX3 GGC Repeat Expansion Underlying Spinocerebellar Ataxia Type 4 has a Common Ancestral Founder.

The identification of a heterozygous exonic GGC repeat expansion in ZFHX3 underlying spinocerebellar ataxia type 4 (SCA4) has solved a 25-year diagnostic conundrum. We used adaptive long-read sequencing to decipher the pathogenic expansion in the index Utah family and an unrelated family from Iowa of Swedish ancestry. Contemporaneous to our discovery, other groups identified the same repeat expansion in affected individuals from Utah, Sweden, and Germany, highlighting the current pivotal time for detection of novel repeat expansion disorders. Given that the pathogenic repeat expansion is rare on a population level, we proposed a common ancestor across all families. Here, we employed targeted long-read sequencing through adaptive sampling, enriching for the chr16q22 region of interest. Using phased sequencing results from individuals from Utah, Iowa, and Southern Sweden, we confirmed a common ~2000-year-old ancestral haplotype harbouring the repeat expansion. This study provides further insight into the genetic architecture of SCA4. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Multispecies emergence of dual blaKPC/NDM carbapenemase-producing Enterobacterales recovered from invasive infections in Chile.

Carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) represent a significant global threat. The emergence of dual CP-CRE is particularly alarming, as they can potentially compromise the efficacy of newer antibiotics, further decreasing therapeutic alternatives. Herein, we report the emergence of multiple species of CP-CRE recovered from invasive infections in Chile that simultaneously harbor blaKPC and blaNDM and provide an in-depth genomic characterization of these worrisome pathogens. We collected carbapenem-resistant Enterobacterales (CRE) isolates from invasive infections over a 4-year period, across 11 healthcare centers in Chile. Bacterial species and the presence of carbapenemase genes were confirmed using MALDI-TOF and PCR assays, respectively. Antimicrobial susceptibility testing was conducted through disk diffusion and broth microdilution methods. Dual CP-CRE isolates were subjected to short- and long-read whole genome sequencing to perform a detailed genomic characterization of the isolates and of the mobile genetic elements harboring the enzymes. From a total of 1,335 CRE isolates, we observed an increase in the prevalence of CP-CRE, from 11% in 2019 to 38% in 2022. A total of 11 dual CP-CRE isolates were recovered, all of them harboring blaKPC and blaNDM. Species corresponded to Escherichia coli (n = 6), Klebsiella pneumoniae (n = 2), Klebsiella oxytoca (n = 2), and Citrobacter freundii (n = 1). Dual CP-CRE isolates exhibited resistance to all tested β-lactams except for cefiderocol. The blaKPC and blaNDM encoding genes were located on independent plasmids. Platforms harboring blaKPC were diverse and included IncN, IncF, and IncFIB plasmids. In contrast, blaNDM-7 was only found on fairly conserved IncX3 plasmids. We report that a rapid increase of CP-CRE in Chile, alongside with the emergence of multiple bacterial species of CP-CRE co-harboring blaKPC-2/3 and blaNDM-7, underscores a critical public health challenge. Our data suggest that the dissemination of blaNDM-7 was predominantly facilitated by IncX3 plasmids, whereas the spread of blaKPC involved multiple plasmid backbones. Active surveillance and genomic monitoring are critical to inform public policy and curtail the spread of these highly resistant pathogens.

Rearrangements of viral and human genomes at human papillomavirus integration events and their allele-specific impacts on cancer genome regulation.

Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer. To resolve genome dysregulation associated with HPV integration, we performed Oxford Nanopore long-read sequencing on 72 cervical cancer genomes from an Ugandan dataset that was previously characterized using short-read sequencing. We found recurrent structural rearrangement patterns at HPV integration events, which we categorized as: del(etion)-like, dup(lication)-like, translocation, multibreakpoint, or repeat region integrations. Integrations involving amplified HPV-human concatemers, particularly multibreakpoint events, frequently harbored heterogeneous forms and copy numbers of the viral genome. Transcriptionally active integrants were characterized by unmethylated regions in both the viral and human genomes downstream from the viral transcription start site, resulting in HPV-human fusion transcripts. In contrast, integrants without evidence of expression lacked consistent methylation patterns. Furthermore, whereas transcriptional dysregulation was limited to genes within 200 kilobases of an HPV integrant, dysregulation of the human epigenome in the form of allelic differentially methylated regions affected megabase expanses of the genome, irrespective of the integrant's transcriptional status. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.

Genetic Analysis and Reproductive Interventions for Two Rare Families Affected by Severe Haemophilia A.

Haemophilia A (HA) is a rare bleeding disorder caused by variants in F8. Although traditional mutational analyses have identified numerous pathogenic variants, the aetiology of HA in certain patients remains unclear. Furthermore, female patients with severe HA are rare. To investigate the molecular defects underlying severe HA in two patients and provide personalised reproductive interventions for their families. Two patients diagnosed with severe HA without other clinical phenotypes were enrolled in the study. A combination of whole-exome sequencing, real-time quantitative polymerase chain reaction and long-read sequencing (LR-sequencing) was performed to reveal the molecular defects of them, followed by the application of different reproductive intervention strategies. Proband 1, a 29-year-old man with FVIII activity of 0.8%, did not exhibit common F8 variants, including Inv1 or Inv22, in the coding region. However, he carried a rare maternal novel inversion on ChrX:154148973_154170321, spanning approximately 21.345Kbp, with breakpoints in introns 13 and 14 of F8. Finally, the couple of Proband 1 opted for assisted reproductive technology using preimplantation genetic testing and successfully conceived. Proband 2, a 20-year-old female with severe HA and FVIII activity of 0.6%, carried inv22 of F8. Further investigation combining whole exome sequencing (WES) and pedigree analysis revealed that she carried a maternal cross-deletion encompassing exons 1-22 of F8, FUNDC2, BRCC3 and CLIC2, along with a de novo missense variant c.5852T>C (p.Leu1951Ser) on her paternal X-chromosome. Chromosome X-inactivation (XCI) analysis demonstrated a highly skewed inactivation of the maternal X chromosome, with a ratio of 98:2. Subsequently, prenatal diagnosis confirmed that the third child in this family did not carry any of the F8 variants present in Proband 2. Our findings provide novel insights into the genetic aetiology of HA and emphasise the importance of a definitive diagnosis in guiding genetic counselling and personalised reproductive interventions for affected individuals and their families.

Long-read sequencing sheds light on key bacteria contributing to deadwood decomposition processes

BackgroundDeadwood decomposition is an essential ecological process in forest ecosystems, playing a key role in nutrient cycling and carbon sequestration by enriching soils with organic matter. This process is driven by diverse microbial communities encompassing specialized functions in breaking down organic matter, but the specific roles of individual microorganisms in this process are still not fully understood.ResultsHere, we characterized the deadwood microbiome in a natural mixed temperate forest in Central Europe using PacBio HiFi long-read sequencing and a genome-resolved transcriptomics approach in order to uncover key microbial contributors to wood decomposition. We obtained high quality assemblies, which allowed attribution of complex microbial functions such as nitrogen fixation to individual microbial taxa and enabled the recovery of metagenome-assembled genomes (MAGs) from both abundant and rare deadwood bacteria. We successfully assembled 69 MAGs (including 14 high-quality and 7 single-contig genomes) from 4 samples, representing most of the abundant bacterial phyla in deadwood. The MAGs exhibited a rich diversity of carbohydrate-active enzymes (CAZymes), with Myxococcota encoding the highest number of CAZymes and the full complement of enzymes required for cellulose decomposition. For the first time we observed active nitrogen fixation by Steroidobacteraceae, as well as hemicellulose degradation and chitin recycling by Patescibacteria. Furthermore, PacBio HiFi sequencing identified over 1000 biosynthetic gene clusters, highlighting a vast potential for secondary metabolite production in deadwood, particularly in Pseudomonadota and Myxococcota.ConclusionsPacBio HiFi long-read sequencing offers comprehensive insights into deadwood decomposition processes by advancing the identification of functional features involving multiple genes. It represents a robust tool for unraveling novel microbial genomes in complex ecosystems and allows the identification of key microorganisms contributing to deadwood decomposition.

Whole-Genome Resequencing and Evolutionary Analysis of Wild Morel Mushroom Morchella sp.

Morels (Morchella sp.) are important edible fungi cultivated mainly in China. Although the relevant culture technology for Morchella is now fundamentally mature, it is limited to the Elata and Rufobrunnea clades, and the artificial culture technology for the Esculenta clade, which also has economic value, has not been extensively studied. In this study, we selected a wild morel belonging to the Esculenta clade as the research material and performed de novo sequencing and assembly of the Morchella sp. (Mosp) genome using second- and third-generation sequencing. The whole-genome size of Mosp was 55.17 Mb with a contig N50 of 1.89 Mb, and the GC content was 47.49%. A total of 10,896 protein-coding genes were identified. The non-coding RNA prediction results showed that there were 329 tRNAs, 65 rRNAs, and 37 snRNAs in the Mosp genome. The functional annotation of the Mosp genes showed that most of the genes were related to the reproductive and metabolic processes of the cells and participated in nutrient digestion, absorption, utilization, and catabolism in morels. There was a high degree of repetition (21.58%) in the Mosp genome, and the sizes of the DNA transposons and the long terminal repeats were 0.55 Mb and 5.85 Mb, respectively. The phylogeny analysis showed that Mosp clusters together with four other Morchella species: Morchella importuna, Morchella conica, Morchella sextelata, and Morchella snyderi. Molecular dating indicated that the differentiation of Mosp and the black morels group occurred about 147.0 million years ago (MYA). In addition, the evolutionary analysis showed that 296 gene families were contracted and 96 gene families were expanded in Mosp versus the related morel species. The results of this study provide new insights into the genome evolution of Mosp and lay the foundation for future in-depth research into the molecular biology and breeding of the genus Morchella.

Landscape of alternative splicing and polyadenylation during growth and development of muscles in pigs

Alternative polyadenylation (APA) is emerging as a post-transcriptional regulatory mechanism, similar as that of alternative splicing (AS), and plays a prominent role in regulating gene expression and increasing the complexity of the transcriptome and proteome. We use polyadenylation selected long-read isoform sequencing to obtain full-length transcript sequences in porcine muscles at five developmental stages. We identify numerous novel transcripts unannotated in the existing pig genome, including transcripts mapping to known and unknown gene loci, and widespread transcript diversity in porcine muscles. The top 100 most isoformic genes are mainly enriched in Gene Ontology terms related to muscle growth and development. It is revealed that intron retention/exon inclusion and the usage of distal polyadenylation site (PAS) are associated with ageing through analyzing changes of AS and PAS during muscle development. We also identify developmental changes in major transcripts and major PASs. Furthermore, genes/transcripts important for muscle development are identified. The results confirm the importance of AS and APA in pig muscles, substantially increasing transcriptional diversity and showing an important mechanism underlying gene regulation in muscles.

High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes.

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both mRNA and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using HCR Flow-FISH for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to characterize full-length mRNA isoforms and observe remarkable uniformity of mRNA isoforms from the transgenic units. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for comparing native and synthetic gene regulation and for building more robust transgenic systems.