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PubMed2026

Chromosome-Level Reference Genome of the Desert Night Lizard Xantusia vigilis.

We present a reference-quality genome assembly for the desert night lizard (Xantusia vigilis). The night lizards (Xantusiidae) are a family of small-bodied lizards found in North America (Xantusia), Central America (Lepidophyma), and Cuba (Cricosaura). The night lizard family has an independent evolutionary history of at least 80 million years from its sister taxa within Scincoidea. The Xantusiids have several unique ecological, behavioral and evolutionary characteristics. For instance, the family contains the only squamate species that form diploid, unisexual, parthenogenic lineages. In addition, most night lizards are viviparous and form stable kin groups that are maintained over multiple years, an unusual life history strategy among lizards. Combining PacBio long-read sequencing, Hi-C, and RNAseq data we developed a reference-quality genome for the desert night lizard, X. vigilis. We assembled a complete mitochondrion and ~ 2.2 Gb nuclear genome, with 20 scaffolds that correlate in size to the X. vigilis karyotype. In addition, we found that X. vigilis chromosome 1 aligns with gene content of both of macrochromosome 1 and microchromosome 9 from a genome assembly of a species in the sister family Cordylidae (Hemicordylus capensis).

Xantusia↗
PubMed2026

Reference genome of the Californian trapdoor spider Aptostichus stephencolberti Bond 2008 (Araneae: Mygalomorphae: Euctenizidae).

We present a reference genome assembly for the trapdoor spider Aptostichus stephencolberti. This species, described in 2008, is endemic to the highly fragmented coastal dune habitats of Northern California from Monterey to the San Francisco Bay Area. Trapdoor spiders are ideal taxa for landscape scale genomic studies owing to their extreme site fidelity and limited dispersal capabilities; these same characteristics make them prone to extinction. Genomic studies of species like A. stephencolberti can reveal novel areas of endemism and high conservation value that may not be evident in species with wider ranges and greater dispersal capabilities. As part of the California Conservation Genomics Project, we constructed the A. stephencolberti reference genome from high quality long-read sequences, scaffolded with proximity ligation Omni-C data. The primary assembly comprises 551 scaffolds spanning 3.63 Gbp, a scaffold N50 of 62.2 Mbp and BUSCO completeness of 95.6%. We estimate 52 chromosomes yet find no (TTAGG)n telomer repeats. Expanding the telomeric repeat search finds an ancestral loss of the repeat from all spiders. Automated annotation using the NCBI refseq pipeline and RNAseq data from whole adults finds 14,067 genes with a BUSCO annotation completeness of 95.56%. Repeat annotation identified 77% of the genome to be interspersed repeats. This resource, the first for family Euctenizidae will facilitate future study and resulting conservation actions of A. stephencolberti and other Aptostichus sp. populations associated with the rapidly changing California coastal dune ecosystem.

Aptostichus stephencolberti↗
PubMed1980

Cloning of influenza cDNA ino M13: the sequence of the RNA segment encoding the A/PR/8/34 matrix protein.

A strategy has been developed for sequencing the single-stranded RNA genes of influenza virus. Restriction fragments derived from double-stranded cDNA copies of total influenza RNA were cloned into the bacteriophage M13mp2 and sequenced by the dideoxy technique. Sequences were extended and overlapped by using the virion RNA as template and priming with small restriction fragments. In the course of this strategy, the nucleotide sequence of segment 7 (1027 nucleotides) was completed and provides the primary structure of the matrix protein (27, 861 daltons). In addition, there is a second long reading frame which partly overlaps the reading frame of the matrix protein.

Base Sequence↗
PubMed2005

Multiplexed discovery of sequence polymorphisms using base-specific cleavage and MALDI-TOF MS.

The completion of the Human Genome Project provides researchers with a reference sequence that covers about 99% of the gene-containing regions and is more than 99.9% accurate. Sequence drafts and completed sequences for several other species are also available to researchers worldwide. The ongoing effort to provide more and more genomic reference information now enables the detection of deviations from this 'genetic blueprint'. Comparative sequencing projects will play a major role in elucidating the meaning of the genetic code and in establishing a correlation between genotype and phenotype. As part of this effort, a number of projects will focus on distinct functional aspects, like resequencing of exons or HLA determining regions. Typically these target regions are short in length and their analysis does not require long read length. To find an efficient solution for these applications, we developed a novel method that allows simultaneous analysis of multiple independent target regions (Multiplexed Comparative Sequence Analysis) by employing base-specific cleavage biochemistry and MALDI TOF-MS analysis.

Humans↗
PubMed2026

A novel allele of Sh1 facilitates the development of waxy-sweet corn from waxy corn.

Waxy corn and sweet corn represent 2 major classes of fresh-eating corn, each with distinct sensory attributes and nutritional compositions. Developing a new variety that combines both waxy and sweet traits would address rising consumer demand and expand new market potential. From a fast neutron-mutagenized population of the waxy corn inbred line HB522, we isolated a novel mutant, designated as wx-sweet, whose kernels simultaneously exhibit waxy and sweet characteristics at the milk-filling stage. Through bulked segregant analysis combined with fine mapping, we mapped the causal locus to SHRUNKEN1 (Sh1) on chromosome 9, which was confirmed by an allelism test with a characterized Mu-insertion allele of Sh1. A 7,227-bp Copia-type long terminal repeat retrotransposon insertion was identified in exon 2 of Sh1 in the wx-sweet mutant by long-read sequencing. Consistently, the novel sh1 allele significantly reduced sucrose synthase activity. Genetic and physiological analyses demonstrate that sh1 and wx1 act synergistically to fine-tune carbohydrate metabolism in the endosperm. Integrated transcriptomic and metabolomic profiling uncover extensive transcriptional reprogramming and redirected metabolic flux, leading to substantial accumulation of sucrose and a range of oligosaccharides. These metabolic shifts underlie the unique simultaneous dual waxy-sweet texture in fresh-eating wx-sweet kernels. In summary, our work not only provides valuable genetic resources for breeding next-generation fresh-eating corn but also, for the first time, elucidates the molecular mechanism by which the sh1 and wx1 mutations cooperatively shape the waxy-sweet endosperm phenotype.

Zea mays↗
PubMed2026

From genotype to phenotype: understanding the genetic basis of autism.

PURPOSE OF REVIEW: This paper covers some of the key findings on the topic of genetic influences in autism over the last 12-18 months, which consist of significant conceptual shifts and new insights from recent technological advances. RECENT FINDINGS: Autism is a highly heritable condition, with significant heterogeneity of the genes that influence the development of this condition. The heterogeneity of autism, and multiple pathways contributing to the development of an autistic phenotype, create challenges in our understanding, diagnosis, and management of this condition.Recent studies of common genetic variation and polygenic risk scores have focussed on resolving phenotypic heterogeneity and identifying meaningful autism subtypes. Rare-variant discovery has expanded across ancestries, the X chromosome, noncoding loci, structural variants, and tandem repeats, aided by long-read and pangenome-informed sequencing. Single-cell multiomics, spatial perturbation methods and human organoid models have connected genetic variation to cell-type-specific and developmental phenotypes, while also revealing substantial mutation-specific effects and methodological sensitivity. Genetic testing increasingly provides aetiological diagnoses and informs medical surveillance. Recent developments also illustrate the therapeutic potential of gene-first approaches for selected monogenic neurodevelopmental disorders. SUMMARY: Recent developments have expanded our understanding of the way the genetic basis of autism manifests phenotypically.

autism↗
PubMed2025

Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair.

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first to be explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from seventeen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, and Hi-C, and karyotyping. In future publications, these data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic mutations. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

Journal Article↗
PubMed2025

The human IG heavy chain constant gene locus is enriched for large structural variants and coding polymorphisms that vary among human populations.

The human immunoglobulin heavy chain constant (IGHC) domain of antibodies (Ab) is responsible for effector functions critical to immunity. This domain is encoded by genes in the IGHC locus, where descriptions of genomic diversity remain incomplete. We utilized long-read sequencing to build an IGHC haplotype/variant catalog from 105 individuals of diverse ancestry. We discovered uncharacterized single nucleotide variants (SNV) and large structural variants (SVs, n=7), representing new genes and alleles enriched for non-synonymous substitutions, highlighting potential functional effects. Of the 221 identified IGHC alleles, 192 were novel. SNV, SV, and gene allele/genotype frequencies revealed population differentiation, including (i) hundreds of SNVs in African and East Asian populations exceeding a fixation index (FST) of 0.3, and (ii) an IGHG4 haplotype carrying coding variants uniquely enriched in Asian populations. Our results illuminate missing signatures of IGHC diversity and establish a new foundation for investigating IGHC germline variation in Ab function and disease.

Journal Article↗
PubMed2025

Perplexity as a Metric for Isoform Diversity in the Human Transcriptome.

Long-read sequencing (LRS) has revealed a far greater diversity of RNA isoforms than earlier technologies, increasing the critical need to determine which, and how many, isoforms per gene are biologically meaningful. To define the space of relevant isoforms from LRS, many existing analysis pipelines rely on arbitrary expression cutoffs, but a single threshold cannot accommodate the broad variability in isoform complexity across genes, cell-types, and disease states captured by LRS. To address this, we propose using perplexity-an interpretable measure derived from entropy-that quantifies the effective number of isoforms per gene based on the full, unfiltered isoform ratio distribution. Calculating perplexity for 124 ENCODE4 PacBio LRS datasets spanning 55 human cell types, we show that it provides intuitive assessments of isoform diversity and captures uncertainty across genes with varying complexity. Perplexity can be calculated at multiple gene regulatory levels-from transcript to protein-to compare how isoform diversity is reduced across stages of gene expression. On average, genes have an ORF-level perplexity of 2.1, indicating production of two distinct protein isoforms. We extended this analysis to evaluate expression variation across tissues and identified 4,593 ORFs across 3,102 genes with moderate to extreme tissue-specificity. We propose perplexity as a consistent, quantitative metric for interpreting isoform diversity across genes, cell types, and disease states. All results are compiled into a community resource to enable cross-study comparisons of novel isoforms.

Journal Article↗
PubMed2025

A complete and near-perfect rhesus macaque reference genome: lessons from subtelomeric repeats and sequencing bias.

A truly complete, telomere-to-telomere (T2T), and error-free reference genome remains a foundational resource-and long-standing goal-for unbiased comparative and functional genomics. While recent T2T assemblies of humans and other primates have made substantial progress, most still contain thousands of base-level errors, particularly within highly repetitive regions. Here, we present T2T-MMU8v2.0, a near-perfect T2T assembly of the rhesus macaque (Macaca mulatta), representing the highest base-level accuracy reported in a primate genome to date. By employing an optimized ONT-only assembly strategy, we identify subtelomeric satellite-rich regions as the principal bottleneck to improving assembly quality, owing to technological biases in long-read platforms and limitations in current hybrid assembly frameworks. We discover 268 previously unannotated repeat families and resolve ~8 Mbp of SATR satellite arrays, with over 99-fold enrichment in historically misassembled subtelomeric regions. These satellites form four distinct genomic architectures, each with unique SATR satellite composition, segmental duplication organization, and epigenetic signatures, distinct from the subtelomeric architectures observed in hominid genomes. Notably, in contrast to the largely gene-poor subtelomeric regions in African hominids, the SATR architectures in macaques harbor 58 actively transcribed genes, supported by open chromatin and expression data, suggesting gene innovation within these repetitive regions. Functionally, T2T-MMU8v2.0 improves read mappability and accuracy across sequencing platforms, and results in a 19% improvement of transcription start site enrichment scores and 5,821 additional chromatin accessibility peaks on average, thereby enhancing variant detection, regulatory annotation, and transcriptomic resolution in population genetics or single-nucleus studies. Together, this work establishes a new benchmark for genomics, offers a roadmap for resolving complex repetitive regions, and reveals previously unrecognized features of subtelomeric genome structure and evolution.

Journal Article↗
PubMed2025

Genomic resources for comparative analyses of obligate avian brood parasitism.

Examples of convergent evolution, wherein distantly related organisms evolve similar traits, including behaviors, underscore the adaptive power of natural selection. In birds, obligate brood parasitism, and the associated loss of parental care behaviors, has evolved independently in seven different lineages, though little is known about the genetic basis of the complex suite of traits associated with this rare life history strategy. We generated genome assemblies for ten brood parasitic species plus eight species representatives of their parental/nesting outgroups. This includes nine long-read chromosome-level assemblies, with scaffold N50 sizes ranging from 38.1 to 72.6 MB, and gene representation completeness measures >97%. Leveraging this new catalog of avian genomes, we constructed clade-level alignments that reveal variation in chromosomal synteny, provide first-time or improved annotations of protein-coding and non-coding genes, and define cross-species ortholog reference sets. We also refine estimates for the timing of the seven independent origins of brood parasitism, ranging from recent events such as 1.6 to 4.5 million years ago in Molothrus cowbirds to much earlier origins over 30 million years ago in two of the three cuckoo lineages. These genomic resources lay the foundation for investigating the genetic and genomic underpinnings of brood parasitism, including the loss of parental care, shifts in mating systems, perhaps resulting in heightened sperm competition, elevated annual fecundity, improved spatial cognition related to nest-finding, and the diverse adaptations shaped by intense coevolution with host species.

assemblies↗
PubMed2025

Genome Report: De novo genome assembly of the greater Bermuda land snail, Poecilozonites bermudensis (Mollusca: Gastropoda), confirms ancestral genome duplication.

Poecilozonites bermudensis, the greater Bermuda land snail, is a critically endangered species and one of only two extant members in its genus. These snails are one of Bermuda's few endemic animal clades and their rich fossil record was the basis for the punctuated equilibria model of speciation. Once thought extinct, recent conservation efforts have focused on the recovery of the species, yet no genomic information or other molecular sequences have been available to inform these initiatives. We present a high-quality, annotated genome for P. bermudensis generated using PacBio long read and Omni-C short read sequencing. The resulting assembly is approximately 1.36 Gb with a scaffold N50 of 44.t Mb and 31 chromosome-length scaffolds. Nearly 43 percent of the genome was identified as repeat content. This assembly will serve as a resource for the conservation and study of P. bermudensis, and its only close extant and also critically endangered relative, P. circumfirmatus. Additionally, this genome adds to the growing body of data needed for a more complete understanding of gastropod evolution and for evolutionary processes in general.

Annotation↗
PubMed2025

Discovery of additional ancient genome duplications in yeasts.

Whole genome duplication (WGD) has had profound macroevolutionary impacts on diverse lineages1,2, preceding adaptive radiations in vertebrates3-5, teleost fish6,7, and angiosperms8,9. In contrast to the many known ancient WGDs in animals10,11 and especially plants12-14, we are aware of evidence for only four in fungi15,16. The oldest of these occurred ~100 million years ago (mya) and is shared by ~60 extant Saccharomycetales species17,18, including the baker's yeast Saccharomyces cerevisiae (Fig. 1). Notably, this is the only known ancient WGD in the yeast subphylum Saccharomycotina. The dearth of ancient WGD events in fungi remains a mystery15. Some studies have suggested that fungal lineages that experience chromosome19 and genome15 duplication quickly go extinct, leaving no trace in the genomic record, while others contend that the lack of known WGD is due to an absence of data15,16. Under the second hypothesis, additional sampling and deeper sequencing of fungal genomes should lead to the discovery of more WGD events. Coupling hundreds of recently published genomes from nearly every described Saccharomycotina species with three additional long-read assemblies, we discovered three novel WGD events. While the functions of retained duplicate genes originating from these events are broad, they bear many similarities to the well-known WGD that occurred in the Saccharomycetales17. Our results suggest that WGD may be a more common evolutionary force in fungi than previously believed.

Convergent Evolution↗
PubMed2025

Quantifying HLA transcripts by genotype in chimeric mixtures at single-cell resolution.

Gene products from the highly variable major histocompatibility locus, including HLA, are essential for self-recognition and immune surveillance of malignancy. Following allogeneic hematopoietic cell transplantation (alloHCT), genetic and epigenetic alterations in HLA can drive disease recurrence, making precise HLA assessment critical for determining future therapy. However, current methods lack the sensitivity to quantify HLA transcripts at the single-cell level, limiting their clinical utility. We introduce scrHLA-typing, a novel technique that accurately identifies and quantifies HLA transcripts in single cells using long-read sequencing. When applied to samples from patients with post-transplant relapse, scrHLA-typing successfully detected HLA allele-specific expression, across a range of levels of donor-recipient chimerism, at clinically actionable levels. By characterizing allele expression in residual leukemia cells, our assay identified differences in expression patterns among patients. This capability highlights scrHLA-typing's potential to improve risk stratification and guide the selection of appropriate salvage therapies, enhancing personalized treatment strategies after relapse.

Journal Article↗
PubMed2025

Candida glabrata replicating within macrophages experiences amino acid deprivation, DNA damage, and chromosome instability.

Macrophages, the central players of innate immunity, control invading microbes by encapsulating them inside the phagosome, a nutrient-poor, reactive oxidant species-rich organelle. Nevertheless, some microbes, including the opportunistic yeast pathogen Candida glabrata, noted for its karyotype diversity, rapid evolution of antifungal drug resistance, and lack of meiosis, can survive and even replicate inside macrophages. However, it is not fully understood how C. glabrata responds to macrophage engulfment, and it is unknown how this presumably DNA-damaging environment influences the pathogen's genome stability. In this study, we used comparative transcriptomics to identify amino acid starvation and DNA damage as conditions eliciting C. glabrata responses most similar to macrophage engulfment. Consistent with this, we found that C. glabrata intra-macrophage survival and replication require master regulator of amino acid biosynthesis GCN4 and functional DNA double-strand break repair. Furthermore, comet assays provided the first direct evidence for increased DNA breaks in intra-macrophage yeast, and pulse-field gel electrophoresis showed that chromosomal alterations occur frequently in macrophage-passaged C. glabrata. Interestingly, these alterations could not be resolved by long read DNA sequencing, suggesting that they involved highly complex repetitive regions. Finally, we identified several point mutations emerging during macrophage passaging and showed that among them, a frameshift in RME1 (repressor of meiosis in Saccharomyces cerevisiae), increased C. glabrata intra-macrophage fitness. Together, these analyses point to amino acid deprivation, reveal elevated DNA breakage and chromosome instability, and raise intriguing questions about the role of meiotic gene orthologs in C. glabrata persisting and replicating within macrophages.

Journal Article↗
PubMed2025

L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements.

LINE-1 retrotransposition is common in human cancers and rearrangements at insertion sites can contribute to cancer-driving oncogene amplifications and promote genome instability. However, the mechanisms underlying rearrangements of L1 retrotransposition intermediates are poorly understood. To address this gap, we developed GFP-based recombination reporter assays to study the formation of L1 retrotransposition-mediated rearrangements. Using these reporters combined with long-read sequencing approaches, we find that L1 retrotransposition intermediates can recombine with distal DNA breaks to generate chromosomal rearrangements. We also find that two distinct L1 insertion intermediates can recombine with each other to generate chromosomal rearrangements. Both types of rearrangements depend on L1-encoded ORF2p endonuclease and reverse transcriptase activities. Using these reporters, we discover that L1 retrotransposition-mediated rearrangements are robustly induced when the recombining sequences share homology and that their formation requires the homologous recombination factor BRCA1. Given the repetitive nature of our genome, these findings highlight the risk of L1 insertion intermediates becoming substrates for aberrant recombination and promoting genome instability.

L1 retrotransposons↗
PubMed2025

Elimination of myotonia improves myopathy in a muscleblind knockout model of myotonic dystrophy.

A cardinal sign of myotonic dystrophy type 1 (DM1) is slow of muscle relaxation after voluntary contraction known as myotonia. Myotonia results from mis-regulated splicing of chloride channel 1 (ClC-1), leading to loss of channel function and runs of involuntary action potentials in muscle fibers. Heralding the onset of weakness, myotonia is often the first symptom of DM1, and raising the possibility that muscle hyperexcitability promotes the subsequent development of myopathy. We used genome editing to test this possibility by deleting the alternatively spliced and frameshift inducing ClC-1 exon 7a (E7a) in the Mbnl1 knockout model of DM1. Although several ClC-1 exons exhibit mis-regulated splicing in DM1, deletion of this single cryptic exon was sufficient to restore ClC-1 function and eliminate myotonia systemically and permanently. As determined by long-read sequencing, deletion of E7a reduced the frequency of other splicing defects in ClC-1 transcripts, likely as a passive consequence of restoring reading frame and nonsense surveillance. Furthermore, we observed significantly improved muscle force generation, fiber-type distribution, and histology, and partial restoration of the muscle transcriptome, including differential gene expression and alternative splicing, in non-myotonic Mbnl1 knockout mice. These results suggest that E7a inclusion is a lynchpin splice event that contributes to skeletal myopathy, highlighting myotonia as a therapeutic target and an outcome of interest in DM1.

Journal Article↗
PubMed2000

RIKEN integrated sequence analysis (RISA) system--384-format sequencing pipeline with 384 multicapillary sequencer.

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3' end and 5' end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month.

Animals↗