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Ay, F.

Publications and source records attributed to Ay, F..

At least 19 recordsLinked to original sources

An Open Benchmark for Systems Vaccinology: Insights from the CMI-PB Challenges

Systems vaccinology approaches have identified factors affecting vaccine responses in multiple studies, but the ability of computational models to generalize these findings to unseen data remains unclear. We established a community resource to create and compare models predicting B. pertussis booster vaccination responses and put such modeling approaches to the test. We compiled multi-modal experimental training data from three independent cohorts (n=117 individuals), and asked investigators to predict vaccine responses in a cohort of 54 newly recruited individuals using only their pre-booster vaccination data. We benchmarked a total of 107 computational models. Top-performing models were characterized by workflows that prioritized rigorous data preprocessing, robust imputation of missing data, and the use of multi-omics integration or non-linear machine learning. We identified pre-existing antigen-specific antibody titers and baseline monocyte frequencies as the most consistent predictors of post-vaccination immunity, highlighting the dominant role of individual immune setpoints. We established the resulting datasets and evaluation framework as a community resource to advance predictive immunology and facilitate personalized vaccination strategies.

immunology↗

Loss of TET function in T regulatory cells yields ex-Treg cells biased toward T follicular helper cells, causingautoimmune diseases through autoantibody production

T regulatory cells (Treg cells) express the transcription factor FOXP3 and maintain immune homeostasis by attenuating effector responses. Treg cells are prone to lose FOXP3 and convert to pathological ex-Treg cells under conditions of strong or chronic inflammation. One mechanism for loss of FOXP3 expression involves increased DNA methylation of intronic enhancers CNS1 and CNS2 in the Foxp3 locus; these enhancers are maintained in a demethylated state by TET enzymes, 5-methylcytosine (5mC) dioxygenases that generate 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines that are essential intermediates in all pathways of DNA demethylation. We previously showed that FOXP3+ Treg cells from Tet2/3-deficient (Tet2/3 DKO) mice displayed increased methylation of CNS1 and CNS2 and converted to FOXP3-negative ex-Treg cells considerably more efficiently than WT Treg cells. Here we extend our previous analysis of Foxp3-Cre Tet2/3fl/fl mice. We classified the mice as DKO- moderate or DKO-severe based on the total number of leukocytes in the spleen and peripheral lymph nodes and investigated the phenotypic and molecular basis for the progressive inflammation occurring in these mice. RNA-seq as well as histological and immunocytochemical analyses showed a striking expansion of T follicular helper (Tfh) cells and plasma cells in Tet2/3 DKO-severe mice. RNA-seq analyses also revealed increased induction of interferon-stimulated genes (ISGs) in CD4+ FOXP3- T cells from these mice, and single-cell (sc) RNA-seq analyses suggested strongly that this was due to skewed differentiation of both Tet2/3 DKO FOXP3+ Treg cells and Tet2/3 DKO FOXP3- ex-Treg cells into Tfh-like cells. Base-resolution "6-base" sequencing showed the expected loss of 5hmC and increased 5mC in Tfh cells purified from Tet2/3 DKO-severe mice, and suggested that the observed bias in gene expression patterns could arise both from a direct increase in methylation of essential enhancers stemming from TET deficiency, or because methylation interfered with binding of methylation-sensitive transcriptional regulators including CTCF.

immunology↗

The interferon response signature links to increased expression of transposable elements in neutrophils of systemic lupus erythematosus patients

BackgroundSystemic lupus erythematosus (SLE) is a spontaneous systemic auto-immune condition for which the inciting factors and genetic basis are generally unknown. Although heterogeneous in its manifestations and severity, SLE involves chronic inflammation along with sustained autoantibody production. The root causes and pathophysiology of the inflammation and breaches of tolerance are incompletely understood, but neutrophils are thought to be important elements of the pathophysiology. Type I interferons (IFN) in the bloodstream and an IFN-stimulated gene (ISG) signature in circulating leukocytes, including neutrophils, are common features in many patients. Earlier work has provided evidence of increased levels of transcripts derived from transposable elements (TEs) in peripheral blood cells of SLE patients. Using six leukocyte types, including neutrophils, we tested the correlation of TE expression with disease severity and explored the relationships between increased ISG and TE expression with attention to the genomic locations of the expressed TEs. ResultsWe reanalysed previously published data from neutrophils and other leukocytes of SLE patients sub-divided into ISG-high (termed IFNpos, n=12) and ISG-low (termed IFNneg, n=11) patients in the original study, examining RNA-seq data from B and T lymphocytes, conventional and plasmacytoid dendritic cells (DC), monocytes and PMN of IFNpos and IFNneg SLE patients compared to healthy controls. SLE patients pre-stratified as IFNneg showed no significant increase in TE expression. All IFNpos cell types had similar amounts of total TE-encoded RNA, but among the 6 cell types, PMN had the highest number of differentially expressed TEs and ISGs in IFNpos SLE patients compared to healthy controls. There was a strong correlation between expression of several specific TE families and disease activity assessed at the time of the visit. Most upregulated TEs ([~]80%) were present in introns of upregulated genes, and [~]67% of these were ISGs. By mapping expressed TEs in ISGs, we found that high intronic TE expression correlated strongly with increased ISG expression as well as with splicing alterations in annotated exons flanking expressed TEs. Consistent with autonomous TE expression, upregulated TEs were also observed at intergenic sites distant from annotated genes, perhaps due to weakening of heterochromatin integrity. ConclusionsOur findings show a strong association and suggest mechanistic relationships between increased TE expression and IFN responses in multiple types of leukocytes centrally involved in SLE pathogenesis. Although limited by short-read RNA-seq technology, our analyses support selective upregulation of some TEs independent from the regulation of conventional genes, concurrent with many intron-localized TEs whose expression tracks with ISGs. The data emphasize the need for long-reads sequencing to understand the causes and consequences of high TE expression in SLE and other autoimmune/inflammatory disorders. Important questions include whether TE expression in introns of ISGs and other genes is independently regulated or reflects exonization or partial intron retention, and how frequently it correlates with splicing variations in adjacent exons.

immunology↗

Genomic Language Model for Predicting Enhancers and Their Allele-Specific Activity in the Human Genome

Predicting and deciphering the regulatory logic of enhancers is a challenging problem, due to the intricate sequence features and lack of consistent genetic or epigenetic signatures that can accurately discriminate enhancers from other genomic regions. Recent machine-learning based methods have spotlighted the importance of extracting nucleotide composition of enhancers but failed to learn the sequence context and perform suboptimally. Motivated by advances in genomic language models, we developed DNABERT-Enhancer, a novel enhancer prediction method, by applying DNABERT pre-trained language model on the human genome. We trained two different models, using large collection of enhancers curated from the ENCODE registry of candidate cis-Regulatory Elements. The best fine-tuned model achieved 88.05% accuracy with Matthews correlation coefficient of 76% on independent set aside data. Further, we present the analysis of the predicted enhancers for all chromosomes of the human genome by comparing with the enhancer regions reported in publicly available databases. Finally, we applied DNABERT-Enhancer along with other DNABERT based regulatory genomic region prediction models to predict candidate SNPs with allele-specific enhancer and transcription factor binding activity. The genome-wide enhancer annotations and candidate loss-of-function genetic variants predicted by DNABERT-Enhancer provide valuable resources for genome interpretation in functional and clinical genomics studies.

bioinformatics↗

CTCF directly binds G-quadruplex structures to regulate genome topology and gene expression

DNA G-quadruplexes (G4s) are non-B form secondary DNA structures that are highly conserved across evolution. G4 structures occupy key regulatory sites in the mammalian genomes and are implicated in several cellular processes. However, the mechanisms by which G4s contribute to distinct facets of genome function are not well understood. Here, we conduct a proteomics screen with G4s of diverse topologies to uncover novel G4 binding activities in genomic regulators of nucleosome remodeling, paraspeckle assembly, RNA splicing, and 3D genome organization. Among the most prominent hits, we identify the genomic architectural protein, CTCF, as one of the strongest G4 binders. Building on this discovery, we perform extensive biochemical validation of CTCF-G4 interaction and identify CTCF mutants, with pronounced affinity for G4s over its consensus DNA motif. By implementing well-established approaches and developing new G4 mapping tools, we define a comprehensive catalog of genomic G4s and investigate their association with CTCF binding. Using genetic reconstitution of mouse embryonic stem cells with a G4-specific CTCF mutant, we ascribe genomic functions to CTCF-G4 interactions in regulation of CTCF occupancy, chromatin looping and gene expression. Interestingly, our studies reveal a subset of G4-linked chromatin loop anchors that form "persistent loops" which are retained even upon CTCF depletion. Collectively, our work establishes the G4 binding activity of CTCF and provides new insights into the functional significance of G4 structures.

molecular biology↗

Soffritto: a deep-learning model for predicting high-resolution replication timing

MotivationReplication Timing (RT) refers to the order by which DNA loci are replicated during S phase. RT is cell-type specific and implicated in cellular processes including transcription, differentiation, and disease. RT is typically quantified genome-wide using two-fraction assays (e.g., Repli-Seq) which sort cells into early and late S phase fractions followed by DNA sequencing yielding a ratio as the RT signal. While two-fraction RT data is widely available in multiple cell lines, it is limited in its ability to capture high-resolution RT features. To address this, high-resolution Repli-Seq, which quantifies RT across 16 fractions, was developed, but it is costly and technically challenging with very limited data generated to date. ResultsHere we developed Soffritto, a deep learning model that predicts high-resolution RT data using two-fraction RT data, histone ChIP-seq data, GC content, and gene density as input. Soffritto is composed of a Long Short Term Memory (LSTM) module and a prediction module. The LSTM module learns long- and short-range interactions between genomic bins while the prediction module is composed of a fully connected layer that outputs a 16-fraction probability vector for each bin using the LSTM modules embeddings as input. By performing both within cell line and cross cell line training and testing for five human and mouse cell lines, we show that Soffritto is able to capture experimental 16-fraction RT signals with high accuracy and the predicted signals allow detection of high-resolution RT patterns. AvailabilitySoffritto is available at https://github.com/ay-lab/Soffritto.

bioinformatics↗

nipalsMCIA: Flexible Multi-Block Dimensionality Reduction in R via Nonlinear Iterative Partial Least Squares

MotivationWith the increased reliance on multi-omics data for bulk and single cell analyses, the availability of robust approaches to perform unsupervised analysis for clustering, visualization, and feature selection is imperative. Joint dimensionality reduction methods can be applied to multi-omics datasets to derive a global sample embedding analogous to single-omic techniques such as Principal Components Analysis (PCA). Multiple co-inertia analysis (MCIA) is a method for joint dimensionality reduction that maximizes the covariance between block- and global-level embeddings. Current implementations for MCIA are not optimized for large datasets such such as those arising from single cell studies, and lack capabilities with respect to embedding new data. ResultsWe introduce nipalsMCIA, an MCIA implementation that solves the objective function using an extension to Non-linear Iterative Partial Least Squares (NIPALS), and shows significant speed-up over earlier implementations that rely on eigendecompositions for single cell multi-omics data. It also removes the dependence on an eigendecomposition for calculating the variance explained, and allows users to perform out-of-sample embedding for new data. nipalsMCIA provides users with a variety of pre-processing and parameter options, as well as ease of functionality for down-stream analysis of single-omic and global-embedding factors. AvailabilitynipalsMCIA is available as a BioConductor package at https://bioconductor.org/packages/release/bioc/html/nipalsMCIA.html, and includes detailed documentation and application vignettes. Supplementary Materials are available online.

bioinformatics↗

Loop Catalog: a comprehensive HiChIP database of human and mouse samples

HiChIP enables cost-effective and high-resolution profiling of chromatin loops. To leverage the increasing number of HiChIP datasets, we developed Loop Catalog (https://loopcatalog.lji.org), a web-based database featuring loop calls from 1000+ distinct human and mouse HiChIP samples from 152 studies plus 44 high-resolution Hi-C samples. We demonstrate its utility for interpreting GWAS and eQTL variants through SNP-to-gene linking, identifying enriched sequence motifs and motif pairs, and generating regulatory networks and 2D representations of chromatin structure. Our catalog spans over 4.19M unique loops, and with embedded analysis modules, constitutes an important resource for the field.

bioinformatics↗

Chromatin structure and var2csa - a tango in regulation of var gene expression in the human malaria parasite Plasmodium falciparum?

Over the last few decades, novel methods have been developed to study how chromosome positioning within the nucleus may play a role in gene regulation. Adaptation of these methods in the human malaria parasite, Plasmodium falciparum, has recently led to the discovery that the three-dimensional structure of chromatin within the nucleus may be critical in controlling expression of virulence genes (var genes). Recent work has implicated an unusual, highly conserved var gene called var2csa in contributing to coordinated transcriptional switching, however how this gene functions in this capacity is unknown. To further understand how var2csa influences var gene switching, targeted DNA double-strand breaks (DSBs) within the sub-telomeric region of chromosome 12 were used to delete the gene and the surrounding chromosomal region. To characterize the changes in chromatin architecture stemming from this deletion and how these changes could affect var gene expression, we used a combination of RNA-seq, Chip-seq and Hi-C to pinpoint epigenetic and chromatin structural modifications in regions of differential gene expression. We observed a net gain of interactions in sub-telomeric regions and internal var gene regions following var2csa knockout, indicating an increase of tightly controlled heterochromatin structures. Our results suggest that disruption of var2csa results not only in changes in var gene transcriptional regulation but also a significant tightening of heterochromatin clusters thereby disrupting coordinated activation of var genes throughout the genome. Altogether our result confirms a strong link between the var2csa locus, chromatin structure and var gene expression. AUTHOR SUMMARYMalaria remains one of the deadliest parasite-borne diseases, causing not only over a half million deaths annually, but also infecting hundreds of millions more. Plasmodium falciparum, the protozoan parasite that is responsible for the most virulent form of human malaria, is transmitted to humans by infected female mosquitoes during a blood meal. Due to a growing resistance to all existing antimalarials, there is a need to identify novel targets to design new antimalarial strategies. Our research builds on the growing body of evidence that supports the role of genome organization or chromatin structure within the nucleus in controlling the parasite development as well as virulence factors designed to circumvent the host immune response. This study identifies genes and structural elements within the Plasmodium falciparum genome that are controlled, at least partially, by the expression of a single unique and highly conserved virulence gene.

systems biology↗

Brd2 is dispensable for genome compartmentalization and replication timing

Replication Timing (RT) refers to the temporal order in which the genome is replicated during S phase. Early replicating regions correlate with the transcriptionally active, accessible euchromatin (A) compartment, while late replicating regions correlate with the heterochromatin (B) compartment and repressive histone marks. Previously, widespread A/B genome compartmentalization changes were reported following Brd2 depletion. Since RT and A/B compartmentalization are two of the most highly correlated chromosome properties, we evaluated the effects of Brd2 depletion on RT. We performed E/L Repli-Seq following Brd2 depletion in the previously described Brd2 conditional degron cell line and found no significant alterations in RT after Brd2 KD. This finding prompted us to re-analyze the Micro-C data from the previous publication. We report that we were unable to detect any compartmentalization changes in Brd2 depleted cells compared to DMSO control using the same data. Taken together, our findings demonstrate that Brd2 depletion alone does not affect A/B compartmentalization or RT in mouse embryonic stem cells.

genomics↗

Master transcription factor binding sites are necessary for early replication control element activity

Eukaryotic genomes replicate in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated with potential to drive cell fate transitions, but mechanisms controlling RT remain elusive. We previously identified "Early Replication Control Elements" (ERCEs) necessary for early RT, domain-wide transcription, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs) but, deletions identifying ERCEs were large and encompassed many putative regulatory elements. Here, we show that ERCEs are compound elements whose RT activity can largely be accounted for by multiple sites of diverse master transcription factor binding (subERCEs), distinguished from other such sites by their long-range interactions. While deletion of subERCEs had large effects on both transcription and RT, deleting transcription start sites eliminated nearly all transcription with moderate effects on RT. Our results suggest a model in which subERCEs respond to diverse master transcription factors by functioning both as transcription enhancers and as elements that organize chromatin domains structurally and support early RT, potentially providing a feed-forward loop to drive robust epigenomic change during cell fate transitions.

molecular biology↗

A systems vaccinology resource to develop and test computational models of immunity

Computational models that predict an individuals response to a vaccine offer the potential for mechanistic insights and personalized vaccination strategies. These models are increasingly derived from systems vaccinology studies that generate immune profiles from human cohorts pre- and post-vaccination. Most of these studies involve relatively small cohorts and profile the response to a single vaccine. The ability to assess the performance of the resulting models would be improved by comparing their performance on independent datasets, as has been done with great success in other areas of biology such as protein structure predictions. To transfer this approach to system vaccinology studies, we established a prototype platform that focuses on the evaluation of Computational Models of Immunity to Pertussis Booster vaccinations (CMI-PB). A community resource, CMI-PB generates experimental data for the explicit purpose of model evaluation, which is performed through a series of annual data releases and associated contests. We here report on our experience with the first such dry run for a contest where the goal was to predict individual immune responses based on pre-vaccination multi-omic profiles. Over 30 models adopted from the literature were tested, but only one was predictive, and was based on age alone. The performance of new models built using CMI-PB training data was much better, but varied significantly based on the choice of pre-vaccination features used and the model building strategy. This suggests that previously published models developed for other vaccines do not generalize well to Pertussis Booster vaccination. Overall, these results reinforced the need for comparative analysis across models and datasets that CMI-PB aims to achieve. We are seeking wider community engagement for our first public prediction contest, which will open in early 2024.

systems biology↗

dcHiC: differential compartment analysis of Hi-C datasets

The compartmental organization of chromatin and its changes play important roles in distinct biological processes carried out by mammalian genomes. However, differential compartment analyses have been mostly limited to pairwise comparisons and with a main focus on only the compartment flips (e.g., A-to-B). Here, we introduce dcHiC, which utilizes quantile normalized compartment scores and a multivariate distance measure to identify significant changes in compartmentalization among multiple contact maps. Evaluating dcHiC on three collections of Hi-C contact maps from mouse neural differentiation (n=3), mouse hematopoiesis (n=10) and human LCL cell lines (n=20), we show its effectiveness and sensitivity in detecting biologically relevant differences, including those validated by orthogonal experiments. Across these experiments, dcHiC reported regions with dynamically regulated genes associated with cell identity, along with correlated changes in chromatin states, replication timing and lamin B1 association. With its efficient implementation, dcHiC not only enables high-resolution compartment analysis but also includes a suite of additional features, including standalone browser visualization, differential interaction identification, and time-series clustering. As such, it is an essential addition to the Hi-C analysis toolbox for the ever-growing number of contact maps being generated. dcHiC is freely available at https://github.com/ay-lab/dcHiC, and examples from this paper can be seen at https://ay-lab.github.io/dcHiC.

bioinformatics↗

COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types.

Common genetic polymorphisms associated with severity of COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Here, we assessed the effects of 679 COVID-19-risk variants on gene expression in a wide-range of immune cell types. Severe COVID-19-risk variants were significantly associated with the expression of 11 protein-coding genes, and overlapped with either target gene promoter or cis-regulatory regions that interact with target promoters in the cell types where their effects are most prominent. For example, we identified that the association between variants in the 3p21.31 risk locus and the expression of CCR2 in classical monocytes is likely mediated through an active cis-regulatory region that interacted with CCR2 promoter specifically in monocytes. The expression of several other genes showed prominent genotype-dependent effects in non-classical monocytes, NK cells, B cells, or specific T cell subtypes, highlighting the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.

immunology↗

Sox2-RNA mechanisms of chromosome topological control in developing forebrain

Precise regulation of gene expression networks requires the selective targeting of DNA enhancers. The Evf2 long non-coding RNA regulates Dlx5/6 ultraconserved enhancer(UCE) interactions with long-range target genes, controlling gene expression over a 27Mb region in mouse developing forebrain. Here, we show that Evf2 long range gene repression occurs through multi-step mechanisms involving the transcription factor Sox2, a component of the Evf2 ribonucleoprotein complex (RNP). Evf2 directly interacts with Sox2, antagonizing Sox2-dependent Dlx5/6UCE activation. Evf2 regulates Sox2 binding at key sites, including the Dlx5/6eii shadow enhancer and Dlx5/6UCE interaction sites. Evf2 differentially targets RNP-associated Sox2 protein pools (PPs), redirecting Sox2-PPs to one repressed gene at the expense of the other. Co-regulation of Dlx5/6UCEintrachromosomal interactions by Evf2 and Sox2 reveals a role for Sox2 in chromosome topology. We propose that RNA organizes RNPs in a subnuclear domain, regulating both long-range UCE targeting and activity through Sox2-RNP sequestration and recruitment.

molecular biology↗

Single-cell transcriptomic analysis of SARS-CoV-2 reactive CD4+ T cells

The contribution of CD4+ T cells to protective or pathogenic immune responses to SARS-CoV-2 infection remains unknown. Here, we present large-scale single-cell transcriptomic analysis of viral antigen-reactive CD4+ T cells from 32 COVID-19 patients. In patients with severe disease compared to mild disease, we found increased proportions of cytotoxic follicular helper (TFH) cells and cytotoxic T helper cells (CD4-CTLs) responding to SARS-CoV-2, and reduced proportion of SARS-CoV-2 reactive regulatory T cells. Importantly, the CD4-CTLs were highly enriched for the expression of transcripts encoding chemokines that are involved in the recruitment of myeloid cells and dendritic cells to the sites of viral infection. Polyfunctional T helper (TH)1 cells and TH17 cell subsets were underrepresented in the repertoire of SARS-CoV-2-reactive CD4+ T cells compared to influenza-reactive CD4+ T cells. Together, our analyses provide so far unprecedented insights into the gene expression patterns of SARS-CoV-2 reactive CD4+ T cells in distinct disease severities.

immunology↗

Integrative transcriptomic analysis of SLE reveals IFN-driven cross-talk between immune cells

The systemic lupus erythematosus (SLE) is an incurable autoimmune disease disproportionately affecting women and may lead to damage in multiple different organs. The marked heterogeneity in its clinical manifestations is a major obstacle in finding targeted treatments and involvement of multiple immune cell types further increases this complexity. Thus, identifying molecular subtypes that best correlate with disease heterogeneity and severity as well as deducing molecular cross-talk among major immune cell types that lead to disease progression are critical steps in the development of more informed therapies for SLE. Here we profile and analyze gene expression of six major circulating immune cell types from patients with well-characterized SLE (classical monocytes (n=64), T cells (n=24), neutrophils (n=24), B cells (n=20), conventional (n=20) and plasmacytoid (n=22) dendritic cells) and from healthy control subjects. Our results show that the interferon (IFN) response signature was the major molecular feature that classified SLE patients into two distinct groups: IFN-signature negative (IFNneg) and positive (IFNpos). We show that the gene expression signature of IFN response was consistent (i) across all immune cell types, (ii) all single cells profiled from three IFNpos donors using single-cell RNA-seq, and (iii) longitudinal samples of the same patient. For a better understanding of molecular differences of IFNpos versus IFNneg patients, we combined differential gene expression analysis with differential Weighted Gene Co-expression Network Analysis (WGCNA), which revealed a relatively small list of genes from classical monocytes including two known immune modulators, one the target of an approved therapeutic for SLE (TNFSF13B/BAFF: belimumab) and one itself a therapeutic for Rheumatoid Arthritis (IL1RN: anakinra). For a more integrative understanding of the cross-talk among different cell types and to identify potentially novel gene or pathway connections, we also developed a novel gene co-expression analysis method for joint analysis of multiple cell types named integrated WGNCA (iWGCNA). This method revealed an interesting cross-talk between T and B cells highlighted by a significant enrichment in the expression of known markers of T follicular helper cells (Tfh), which also correlate with disease severity in the context of IFNpos patients. Interestingly, higher expression of BAFF from all myeloid cells also shows a strong correlation with enrichment in the expression of genes in T cells that may mark circulating Tfh cells or related memory cell populations. These cell types have been shown to promote B cell class-switching and antibody production, which are well-characterized in SLE patients. In summary, we generated a large-scale gene expression dataset from sorted immune cell populations and present a novel computational approach to analyze such data in an integrative fashion in the context of an autoimmune disease. Our results reveal the power of a hypothesis-free and data-driven approach to discover drug targets and reveal novel cross-talk among multiple immune cell types specific to a subset of SLE patients. This approach is immediately useful for studying autoimmune diseases and is applicable in other contexts where gene expression profiling is possible from multiple cell types within the same tissue compartment.

genomics↗

Third generation sequencing revises the molecular karyotype for Toxoplasma gondii and identifies emerging copy number variants in sexual recombinants.

Toxoplasma gondii is an obligate intracellular parasite that has a significant impact on human health, especially in the immunocompromised. This parasite is also a useful genetic model for intracellular parasitism given its ease of culture in the laboratory and relevant animal models. However, as for many other eukaryotes, the T. gondii genome is incomplete, containing hundreds of sequence gaps due to the presence of repetitive and/or uncloneable sequences that prevent complete telomere-to-telomere de novo chromosome assembly. Here, we report the first use of single molecule DNA sequencing to generate near complete de novo genome assemblies for T. gondii and its near relative, N. caninum. Using the Oxford Nanopore Minion platform, we dramatically improved the contiguity of the T. gondii genome (N50 of [~]6.6Mb) and increased overall assembled sequence compared to current reference sequences by [~]2 Mb. Multiple complete chromosomes were fully assembled as evidenced by clear telomeric repeats on the end of each contig. Interestingly, for all of the Toxoplasma gondii strains that we sequenced (RH, CTG, IIxIII F1 progeny clones CL13, S27, S21, and S26), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome. This was due to a repeatable and consistent fusion of chromosomes VIIb and VIII. These data were further validated by mapping existing T. gondii ME49 Hi-C data to our assembly, providing parallel lines of evidence that the T. gondii karyotype consists of 13, rather than 14, chromosomes. In addition revising the molecular karyotype we were also able to resolve hundreds of repeats derived from both coding and non-coding tandem sequence expansions. For well-known host-targeting effector loci like rhoptry protein 5 (ROP5) and ROP38, we were also able to accurately determine the precise gene count, order and orientation using established assembly approaches and the most likely primary sequence of each using our own assembly correction scripts tailored to correcting homopolymeric run errors in tandem sequence arrays. Finally, when we compared the T. gondii and N. caninum assemblies we found that while the 13 chromosome karyotype was conserved, we determined that previously unidentified large scale translocation events occurred in T. gondii and N. caninum since their most recent common ancestry.

genomics↗