Biology subjects

Jain, M.

Publications and source records attributed to Jain, M..

At least 19 recordsLinked to original sources

bioRxiv2026

tRNAZAP: Deep learning toolkit for nanopore tRNA signal analysis

tRNA are short, ~76 nucleotide-long, modification-rich molecules that play an important role in protein translation. Nanopore sequencing is the only platform capable of directly sequencing full-length, native tRNA molecules. This capability is enabled by splinted ligation of tRNA-specific adapters. As a result, the measured ionic current signal reflects a composition of both adapter and tRNA sequence features. Despite these advantages, analysis of nanopore tRNA sequencing data poses distinct computational challenges. Existing ionic current-based signal-to-sequence decoding (basecalling) models are optimized for mRNA and perform suboptimally on short, modification-dense RNA such as tRNA. Furthermore, alignment algorithms for reference-guided analysis of short-length reads are not optimized for nanopore reads with lower accuracy than Next-generation sequencing reads. To overcome these limitations, domain-specific computational approaches are required. We present tRNAZAP, a nanopore ionic current signal analysis toolkit designed to enable precise reference-guided sequence alignment. tRNAZAP operates directly on raw ionic current signals and decomposes the analysis into two complementary tasks: 1) segmentation of adapter and tRNA regions, and 2) tRNA classification, both implemented using transformer-based models. The segmentation component isolates the tRNA signal portion and extracts the corresponding nucleotide sequence as the alignment query, while the classification component identifies the tRNA type as the alignment reference without requiring sequence-level information. Together, these outputs allow direct optimal pairwise alignment between the tRNA subsequence of the full query and reference sequences. Trained on in vitro transcribed RNA data and evaluated on yeast tRNA, these models achieved ~98% classification accuracy and intersection-over-union of ~0.98 for segmentation.

bioinformatics↗

bioRxiv2026

Mutation in Ralstonia pseudosolanacearum F1C1 cytochrome p450 homologue impairs virulence through root but not through shoot of tomato plants

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

pathology↗

bioRxiv2026

A strategy for ionic current analysis of nanopore protein readouts

BackgroundNanopore-based protein reading holds promise for single-molecule proteomics, but analyzing the resulting ionic current signals presents substantial computational challenges. Unlike nucleic acids, peptide-associated ionic signals exhibit complex patterns with subtle transitions between amino acids that can be obscured by noise. We developed an integrated computational strategy for processing nanopore protein ionic current signals from synthetic protein constructs containing all 20 amino acids in controlled sequence contexts. We started with denoising to remove artifacts, followed by change point detection using variable-number and fixed-number segmentation methods. ResultsWe employed dynamic time warping barycenter averaging to generate amino acid-specific reference templates. In parallel, we performed classification of amino acid regions using bidirectional LSTM networks trained on segment-level statistical features. Accuracy for 20-way amino acid classification remained limited, while charge-based binary classification reached 93.3% accuracy. Pairwise classification revealed that certain amino acids, particularly negatively charged residues, generate distinctive signatures. We used a profile hidden Markov model to capture sequential structure of protein translocation. ConclusionThis approach provides computational strategies for automated segmentation, classification, and modeling of nanopore peptide translocation ionic current data.

bioinformatics↗

bioRxiv2026

Protective near-germline antibodies to malaria elicited by RTS,S/AS01E vaccination in African infants

Malaria is a leading cause of childhood mortality, yet infant immune responses to vaccination remain poorly understood. Here, we characterize 88 monoclonal antibodies (mAbs) directed against the Plasmodium falciparum circumsporozoite protein cloned from African infants immunized with the RTS,S/AS01E malaria vaccine, identifying potent antiparasitic mAbs exhibiting molecular features resembling those observed in adults and minimal somatic hypermutation. Importantly, germline-reverted variants of four mAbs inducing sterile protection in vivo maintained high binding affinity and Fab-dependent parasite killing. Structural analyses revealed that a few amino acid replacements drive homotypic helical assemblies, fine-tuning parasite inhibition and protective efficacy. Thus, despite maturational constraints of early life, infants can generate potent antimalarial antibodies in a near-germline configuration, informing next-generation vaccine development for this vulnerable group.

immunology↗

bioRxiv2026

Seed Microbiome Transfer Mitigates Intergenerational Dysbiosis, Modulates Plant Defenses and Suppresses Foliar Disease

Antibiotic-induced disruption of plant-associated microbiomes has the potential to alter host health beyond the directly exposed generation, yet whether the effects of dysbiosis are transmitted through the seed microbiome remains unknown. Here, we investigated the intergenerational impacts of streptomycin-induced dysbiosis in tomato (Solanum lycopersicum), demonstrated that seed microbiome transfer (SMT) restores progeny microbiome function and disease resistance, and characterized the underlying physiological and genetic mechanisms. Parental streptomycin exposure altered the composition of progeny rhizosphere bacterial communities, reduced expression of defense-associated genes, and increased susceptibility to Xanthomonas perforans. Suppression of immune gene expression was strongly associated with increased disease severity, indicating that parental dysbiosis impaired progeny plants ability to mount effective immune responses. Transfer of the seed microbiome from healthy plant donors partially restored rhizosphere community composition, reduced disease severity and recovered defense gene expression of three genes. Together, our findings demonstrated that antibiotic exposure microbiome disturbance generates intergenerational legacy effects that influence plant immunity and disease susceptibility and seed microbiome transfer can counteract this dysbiosis across generations.

plant biology↗

bioRxiv2026

SALRR: Scalable Analysis of Long-Read RNA-Seq Enables Comprehensive Transcriptome Profiling in Human Brain

Isoform-resolved transcriptomics is fundamental to decoding the molecular complexity of the human brain, yet population-scale long-read RNA sequencing has remained inaccessible due to labor-intensive library preparation, sensitivity to RNA degradation in postmortem tissue, and the absence of integrated, reproducible analysis pipelines. Here we present SALRR (Scalable Analysis of Long-Read RNA-seq), an integrated wet-lab and computational platform designed to overcome these barriers. Automated ONT long-read cDNA library preparation on the Hamilton Microlab NGS STAR platform reduces hands-on time by 67% and enables 24 libraries per operator per day while maintaining performance across RNA integrity values. A modular, Snakemake-based pipeline performs end-to-end processing from ONT signal data to isoform-level quantification, incorporating SIRV spike-in calibration, multi-stage quality control, and stringent isoform validation. Applied to 10 postmortem frontal cortex samples from the North American Brain Expression Consortium, SALRR identified 31,607 high-confidence isoforms from 10,075 genes, including 8,532 novel splice variants absent from GENCODE v49, and complex splicing events systematically missed by short-read sequencing at neurodegeneration-relevant loci, including GBA1, CCNF, CHCHD10, and TREM2. All protocols and code are openly available, providing a scalable, community-ready framework for isoform-resolved transcriptomics in neurodegeneration, aging, and complex brain disease.

genomics↗

bioRxiv2026

Long-read sequencing maps transposable element variation and its regulatory and epigenetic effects in the human brain

Transposable elements (TEs) are mobile DNA sequences that shape genome architecture and gene regulation, yet their roles in the human brain remain largely unresolved. Short-read sequencing lacks the resolution to accurately map TE insertions, detect associated structural variants, and resolve highly repetitive regions. Here, we leverage long-read whole-genome sequencing to profile germline TE insertions in postmortem brain tissue from two ancestrally diverse cohorts: the North American Brain Expression Consortium (NABEC; European ancestry, n = 205) and the Human Brain Collection Core (HBCC; African and African-admixed ancestry, n = 146). We identified 2,842 and 1,660 high-confidence non-reference insertions in HBCC and NABEC, respectively, spanning Alu, LINE-1, and SVA elements. We then also further characterized complex short tandem repeat and variable number tandem repeat variation within reference SVA and Alu loci. Reference TEs were also found to mediate complex structural variants at loci implicated in brain development and neurodegenerative disease, with several showing ancestry-specific patterns. Integration of bulk RNA-sequencing data identified TE expression quantitative trait loci, including insertions that modulate neuronal gene expression. Single-nucleus RNA sequencing revealed cell-type-specific effects of TE regulation across cortical populations. Long-read methylation profiling further demonstrated age-associated epigenetic regulation of both reference and non-reference Alu elements. As a community resource, we release a catalog of TE insertions, allele frequencies, and ancestry-specific distributions to enable future functional and disease-focused investigations. Together, these findings highlight the widespread regulatory and epigenetic influence of TEs in the human brain and establish long-read sequencing as a powerful approach for uncovering cell-type- and population-specific TE dynamics.

genomics↗

bioRxiv2026

Intact and single-molecule analysis of heparan sulfate

Establishing tools to couple biological processes to a DNA sequence has transformed our ability to monitor life at the molecular scale due to the scalability, flexibility, and low cost of DNA sequencing. Key examples include DNA-protein (ChIP-seq1), RNA-protein (CLIP-seq2), protein-protein (proximity ligation assay3), and Cas-based recording of cellular events4. In contrast, this paradigm has not yet significantly enhanced studies of glycans, which are mostly limited to non-DNA based chemical and biochemical assays. While classical asparagine-linked and serine/threonine-linked glycans can be directly sequenced using mass spectrometry, glycosaminoglycans - notable players in the extracellular matrix - cannot be easily analyzed in their full-length form. Here we introduce HS-nano-seq, a generalized framework to selectively label, process, and detect features of heparan sulfate on a nanopore sequencing platform. Recognizing that heparan sulfate is biochemically analogous to a nucleic acid, we report purification techniques using rapid nucleic acid strategies and conjugation methods to couple DNA adapters, generating HS-DNA chimeras resolved as discrete species by capillary electrophoresis (CE). The CE assay can distinguish features of chain length and sulfation patterns. At the single-molecule level enabled by nanopore sensing, we classify a library of synthetic heparan sulfate standards and demonstrate that nanopore ionic current fingerprints encode sulfation-dependent structural features of individual HS chains. Analysis of intact, cell-derived HS could discriminate features of individual chains with different sulfation patterns, defining the heterogeneity of binding motifs across cell types and how cells organize and program the tethered extracellular matrix. More broadly, HS-nano-seq establishes a framework for achieving full-length readouts of ECM glycopolymers that are amenable to the same biological interrogation as nucleic acids.

biochemistry↗

bioRxiv2026

FALCON: Closed-Loop Multi-Objective Optimization of Lipid Nanoparticles for Cell-Selective mRNA Delivery

Efficient, cell type-selective delivery of genetic payloads remains a central challenge in the development of gene and cell therapies. Lipid nanoparticles (LNPs) offer a versatile delivery platform, but their optimization is hindered by reliance on brute-force screening methods that are laborious, resource-intensive, and focus on single targets. Here, we present FALCON (Framework for Active Learning-driven Compositional Optimization of Nanoparticles), a closed-loop pipeline that leverages iterative screening, surrogate modeling, and multi-objective optimization to accelerate LNP compositional design. In B cell-targeted validation experiments, FALCON-optimized LNPs achieved a 1.8-fold increase in splenic B cell transfection in vivo compared with reference compositions. When optimized for selectivity, FALCON LNPs displayed an 84-fold improvement in selective transfection of splenic B cells over off-target liver populations and enabled spleen-tropic behavior across factorial panels of varying ionizable and helper lipid chemistries. In vaccine studies, these LNPs induced higher IgG2c antibody titers and a more Th1-biased immune profile. FALCON was also deployed to optimize LNPs for myeloid cell-selective delivery, achieving enhanced in vivo selectivity following systemic administration both across and within spleen and liver compartments. Our results establish FALCON as a useful tool for data-driven design of LNP compositions for precision gene delivery.

bioengineering↗

bioRxiv2026

Structure and functional diversity of antibodies targeting the P. falciparum circumsporozoite protein C-terminal domain

The Plasmodium falciparum circumsporozoite protein (PfCSP) is the major surface antigen on Pf sporozoites. WHO-recommended vaccines RTS,S/AS01E and R21/Matrix-M target the PfCSP major repeat region and C-terminal domain (ctCSP). Although multiple studies associated protection with antibody responses to ctCSP, only a few ctCSP-specific monoclonal antibodies (mAbs) have been characterized. Here, crystal structures of 11 Fab-ctCSP complexes reveal how mAbs against the conserved {beta}-epitope region achieve diverse modes of strain-transcending recognition, in contrast to mAbs to the hypervariable -epitope. Consistent with previous studies, ctCSP on sporozoites could be unmasked by mAbs that bind CSP repeats, with unmasking dependent on the mAb fine-specificity and binding mode. In vitro, ctCSP mAbs promoted stronger Fc-receptor signaling, cellular cytotoxicity, and phagocytosis than repeat region mAbs, while mAb combinations targeting distinct PfCSP epitopes modulated Fc-signaling and cellular cytotoxicity. This study provides a rationale for optimization of PfCSP-based immunogens to enhance Fc-mediated contributions to malaria vaccine efficacy.

microbiology↗

bioRxiv2026

Integrating mass spectrometry with Nanopore direct RNA sequencing for de novo modification profiling of bacteriophage MS2

RNA modifications contribute to the regulatory and structural complexity of RNA molecules, influencing key biological processes. Nanopore direct RNA sequencing (DRS) enables the detection of these modifications at single-molecule resolution without chemical conversion. Oxford Nanopore Technologies recently introduced RNA004 sequencing chemistry and the Dorado basecaller, which improves accuracy and enables the identification of eight RNA modifications. However, the reliability of these predictions requires careful validation using orthogonal approaches. Here, we profiled RNA modifications in the MS2 bacteriophage genome using both LC-MS/MS and Nanopore DRS. LC-MS/MS analysis revealed that {Psi} is the predominant modification, present at approximately one site per RNA molecule, while all other modifications were found at low levels (< 0.04 modifications per RNA). In contrast, Dorado-based modification calling not only confirmed {Psi} but also predicted multiple m6A, m5C, 2OMe, and inosine sites not supported by LC-MS/MS data. To refine modification calling, we used in vitro transcribed (IVT) RNA as a negative control and subtracted IVT-derived false positive rates from native RNA predictions. This adjustment reduced overcalling and improved confidence in site-specific predictions. These data demonstrate that while Dorado can detect RNA modifications de novo, its predictions require careful filtering and validation. Our studies of provide a rare benchmark dataset for assessing the ability of transcriptome-wide methods to accurately identify RNA modifications and estimate modification stoichiometry. These findings support the use of orthogonal approaches, including LC-MS/MS and IVT controls, alongside Nanopore sequencing to provide a more reliable and interpretable strategy for studying RNA modification patterns. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/729303v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@637a55org.highwire.dtl.DTLVardef@f48272org.highwire.dtl.DTLVardef@a0465aorg.highwire.dtl.DTLVardef@6af045_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Expression-dependent but strand-independent synonymous single-nucleotide polymorphism in the Escherichia coli chromosome

AbstractMutation in genomes is mainly attributed to replication, though transcription is also known to be mutagenic and is a more frequent event than replication in an organism. Recently, there have been reports regarding genome-wide transcription-induced mutagenesis. However, a distinct demonstration of specific mutation being replication-dependent and/or transcription-dependent in genomes is yet to be established. Here, we studied synonymous single-nucleotide polymorphisms (SNPs) in 2091 individual coding sequences (CDS) in the leading strand (LeS) and the lagging strand (LaS) of the Escherichia coli chromosome by comparing across 157 strains. The frequencies of complementary transitions (ti) and complementary transversions (tv) were compared in each CDS to assess parity violation in the context of strand location and gene expression. The C[->]T and G[->]A exhibited the maximum frequency as well as the most prominent strand inequality as these tis were influenced both by the strand location as well as by the gene expression. Interestingly, inequality between T[->]C and A[->]G was expression-dependent but strand-independent. This was a direct demonstration of strand inequality due to expression but not due to replication. A[->]T and G[->]T tvs were universally more frequent than their complementary T[->]A and C[->]A, respectively. The strand-independent but expression-dependent synonymous SNP inequality in CDS, supports the role of transcription-induced mutagenesis contributing to strand inequality in the E. coli chromosome. Significance StatementMutational patterns in bacterial genomes are shaped by both replication and transcription, but their relative roles remain unclear. By analyzing synonymous SNPs across 157 strains of E. coli, we identify strong strand-specific asymmetry consistent with replication-associated biases. A strong asymmetry between the strands regarding C[->]T and G[->]A ti is observed, as it is influenced by replication asymmetry and expression. We further show that gene expression significantly influences mutation types, with T[->]C ti being enriched in the highly expressed genes and A[->]G ti being enriched in the lowly expressed genes. Notably, A[->]T ti is strand independent but exhibits mild dependency on gene expression, revealing a previously unrecognized mutational pattern. These findings highlight how some SNPs are influenced by transcription but not replication

evolutionary biology↗

bioRxiv2026

Morphology of petiole bending, senescence, epinasty, along with necrotic scarring in tomato leaves infiltrated with virulent Ralstonia pseudosolanacearum

Ralstonia pseudosolanacearum (Rps) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 109 CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::{Omega} mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::{Omega}. It indicated that the necrotic lesion caused in tomato leaves was hrp-dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

pathology↗

bioRxiv2026

A complete human pancreatic cancer genome

Cancer genome sequencing is essential for understanding tumor evolution and advancing precision medicine.1 However, reference gaps and germline variants obscure detection of small and large somatic variants and methylation in repetitive regions.1-3 It is common for tumor cells to gain or lose chromosome arms due to somatic structural changes that occur inside highly repetitive satellite DNA sequences in the centromeres.4 To identify the full spectrum of somatic variants, including complex rearrangements, we construct and curate near-complete, haplotype-resolved assemblies of the most recent common ancestor of an early-passage broadly-consented hypodiploid pancreatic cancer cell line and matched normal tissues. The tumor assembly completely recapitulates all 35 tumor chromosomes observed with karyotyping, with multiple translocation-induced hybrid chromosomes. The hybrid chromosomes contain putative functional dicentric and fused centromeres, nested foldback inversions causing 14 breakpoints with a haplotype switch in a single event, and centromeric satellite tandem duplications up to 136 kbp. Direct comparison of tumor and normal assembly haplotypes uncovers >7,000 variants altering >1 Mbp of sequence in repetitive regions that have been hidden by reference gaps and germline variants. 44 % of somatic small variants change representation because they alter germline variants on GRCh38, impacting mutational signatures and kataegis/omikli clusters. Most somatic LINE insertions originate from two hypomethylated non-reference germline LINE insertions, highlighting their impact on insertion mutation burden. These assemblies demonstrate that centromeric, acrocentric, and telomeric regions conventionally excluded from analysis harbor extensive somatic and epigenetic changes. Resolving complete tumor genomes enables a deeper understanding of cancer structural plasticity and the endpoints of breakage-fusion-bridge cycles. These assembled, curated paired normal-tumor benchmarks will serve as a critical foundation for developing future algorithms to characterize the most intractable regions of cancer genomes.

genomics↗

bioRxiv2026

Pseudouridylation landscape across 42 S. cerevisiae cytosolic tRNA isoacceptors via Nanopore direct RNA sequencing

Pseudouridine is the most abundant RNA base modification due to its prevalence in tRNA and rRNA, where it serves as a key modulator of structure and function. Yet, in the widely used model organism, the budding yeast Saccharomyces cerevisiae, tRNA pseudouridine sites have not been comprehensively annotated. Here, we performed two high-throughput methods established for detecting tRNA pseudouridylation positions: Nanopore direct RNA sequencing (DRS) and 2-bromoacrylamide-assisted cyclization sequencing (BACS). Using DRS, we sequenced cytosolic tRNA from seven pseudouridine synthase (PUS) knockout S. cerevisiae strains, including deletion strains of Pus1, Pus3, and Pus7. Analysis of these data verified thirty-two existing pseudouridine annotations and predicted an additional ten previously unannotated pseudouridine sites. Our analysis also revealed DRS signal changes at several non-uridine sites with the loss of a PUS, including apparent changes in modification abundances at position 37 upon deletion of Pus3. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and primer extension assays, however, indicated no change in the abundance of these modifications with the loss of Pus3, underscoring the need for caution in interpreting DRS-based signal changes. Using BACS, we confirmed the ten DRS-predicted novel sites, while further detecting eleven additional previously unannotated pseudouridine sites. Combining existing modification annotations from the Modomics database with our DRS and BACS datasets, we created a map of all detected pseudouridines --totaling 126 sites, including 21 novel sites--and the enzymes responsible for their catalysis, across the forty-two nuclear-encoded S. cerevisiae tRNA isoacceptors.

molecular biology↗

bioRxiv2026

SPIN: A Scalable Bioinformatics Pipeline for Screening Pathogenicity Related Host-Pathogen Protein INteractions Using AlphaFold3

Traditional experimental approaches have greatly advanced our understanding of protein-protein interactions (PPIs) that govern host susceptibility or resistance. Nonetheless, the molecular characterization of microbial effectors and their cognate host targets remains challenging in many economically important plant pathosystems. AlphaFold3 (AF3) has transformed structural biology by achieving near-experimental accuracy in protein structure and complex prediction. A bioinformatics pipeline SPIN (Screening Pathogenicity-Related Host/Pathogen Protein INteractions) was developed for large-scale prediction of interactions between the host and pathogen-secreted proteins. SPIN integrates three core modules: pathogen and host protein preprocessing using bioinformatics tools (SignalP6.0, OrthoFinder, CD-HIT), AF3-based interaction modeling, supported by automated input generation and output filtering. However, AF3 training bias toward mammalian proteins necessitated careful evaluation in plant systems. Benchmarking against experimentally validated plant PPIs revealed that derivative metrics emphasizing interfacial geometry and residue-level contact (ipSAE and pDockQ) provide superior discrimination compared to native global confidence measures (pTM and ipTM), particularly for proteins with intrinsically disordered regions. A multi-metric confidence scoring framework combining pLDDT, PAE, ipSAE, and pDockQ, improved prediction reliability by enhancing recall and reduced false positives through robust assessment of structural confidence and interface quality. For proof-of-concept, SPIN was applied to examine the molecular landscape underlying two economically important diseases of citrus (Citrus L.) caused by Candidatus Liberibacter asiaticus and Ca. Phytoplasma citri. Both pathogens are phloem-limited and cause distinct symptoms, citrus greening and witches broom, respectively. AF3-predicted interactome data revealed conserved host colonization strategies alongside disease-specific molecular mechanisms, demonstrating the utility of SPIN for dissecting and supporting mechanistic studies in plant-pathogen interactions.

genomics↗

bioRxiv2026

The diploid reference genome of a human embryonic stem cell line

Advances in DNA sequencing and assembly technologies are spurring a shift from haploid reference genomes to sample-specific diploid assemblies. Here, we generated the first telomere-to-telomere (T2T) diploid reference for the widely used human embryonic stem cell (hESC) line, H9 (WAe009-A). This haplotype-resolved assembly is highly accurate with comprehensive annotation of genes, segmental duplications, methylation, and chromatin conformation. Pangenomic and phased-locus inference point to H9s mixed ancestry with a predominant European component. H9-specific genomic features include near-perfect telomeres [~]1.65-fold longer than other T2T assemblies, consistent with telomerase activity during pluripotency; chromosome 17 inversions that can predispose offspring to neurological syndromes; and expansions of ncRNA clusters, with overall genomic stability maintained despite extensive culturing. Mapping multi-omic datasets to the genome, we demonstrate the power of this resource for allele-specific, high-precision transcriptomic, genetic, and epigenetic analyses, with far-reaching implications for human development and disease.

genomics↗

bioRxiv2026

The complete genome of the KOLF2.1J reference iPSC line

While induced pluripotent stem cells (iPSCs) have gained popularity in studying neurodegenerative diseases, the heterogeneity of stem cells used across studies impacts cross-study comparison. The iPSC Neurodegenerative Disease Initiative (iNDI) selected the KOLF2.1J cell line and prioritized its use as a reference standard for studying the effects of pathogenic variants on cell biology due to its stability and neutral neurodegenerative disease genetic risk. This cell line, and its derivatives expressing over 100 variants related to Alzheimers disease, Parkinsons disease, and other neurological diseases, are available for academic and industry access. Current genomic data analyses are limited by the use of a human reference genome that does not capture the complete genetic background of a given iPSC line. While in the future this issue may be partially mitigated by the creation of a comprehensive human pangenome, previous work has shown that generating custom genomes is of value both to characterize the variation present and to serve as a more appropriate genomic reference. Here, we generated and characterized a custom complete genome assembly from KOLF2.1J. Mapping of sequencing reads to a personalized diploid assembly results in more comprehensive mapping compared to traditional linear references (i.e GRCh38). In addition, we provide a comprehensive custom gene annotation along with isoform expression and differential methylation analyses across multiple cell types. The assembly and all additional data is browsable and publicly available. This resource will enable more accurate investigation of the KOLF2.1J cell line and any genomics data generated compared to using traditional generalized references, while also serving as a foundational approach for establishing custom reference assemblies for other high-value iPSC lines.

genomics↗