Biology subjects

Zhao, C.

Publications and source records attributed to Zhao, C..

At least 199 records · Page 11Linked to original sources

bioRxiv2023

KDM2B regulates hippocampal morphogenesis by transcriptionally silencing Wnt signaling in neural progenitors

The hippocampus plays major roles in learning and memory. Similar to other parts of the brain, the development of hippocampus requires precise coordination of patterning, cell proliferation, differentiation, and migration, with both cell-intrinsic and extrinsic mechanisms involved. Here we genetically removed the chromatin-association capability of KDM2B - a key component of the variant Polycomb repressive complex 1 (PRC1) - in the progenitors of developing dorsal telencephalon (Kdm2b{Delta}CxxC) to surprisingly discover that the size of Kdm2b{Delta}CxxC hippocampus, particularly the dentate gyrus, became drastically smaller with disorganized cellular components and structure. Kdm2b{Delta}CxxC mice displayed prominent defects in spatial memory, motor learning and fear conditioning. The differentiation trajectory of the developing Kdm2b{Delta}CxxC hippocampus was greatly delayed, with a significant amount of TBR2-expressing intermediate progenitors stuck along the migratory/differentiation path. Transcriptome and chromatin immunoprecipitation studies of neonatal hippocampi and their progenitors indicated that genes implicated in stemness maintenance, especially components of canonical Wnt signaling, could not be properly silenced by PRC1 and PRC2. Activating the Wnt signaling disturbed hippocampal neurogenesis, recapitulating the effect of KDM2B loss. Together, we unveiled a previously unappreciated gene repressive program mediated by KDM2B that controls progressive fate specifications and cell migration, hence morphogenesis of hippocampus during development. Graphic AbstractLoss of KDM2B-CxxC reduces repressive histone modifications - H2AK119ub and H3K27me3 - on key Wnt signal genes, hence leading to the block of their attenuation over time. Hampered differentiation and migration of hippocampal progenitors leads to hippocampal agenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/525636v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@18fbbdborg.highwire.dtl.DTLVardef@17633f9org.highwire.dtl.DTLVardef@14e36b9org.highwire.dtl.DTLVardef@1eadb8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

CAM5, WRKY53, and TGA5 regulate defense gene expression mediated by the volatile organic compound ethyl vinyl ketone

Plants produce ethyl vinyl ketone (evk) in response to biotic stress, but the evks identification and downstream defense response remain unclear. In this paper, it is predicted by docking for the first time that evk can be recognized by RBOH protein and assist the electron transfer of RBOHD/RBOHF by binding to its FAD or NADPH binding site. Here, we show that evk treatment increased H2O2 and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H2O2 signaling functioned upstream of Ca2+ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca2+-ATPASE, ISOFORM 8)-CML8 (CALMODULIN-LIKE 8) interaction promoted Ca2+ efflux to return Ca2+ levels to the resting state. Evk treatment led to the antagonism of salicylic acid (SA) and jasmonic acid (JA). CALMODULIN 5 (CAM5) positively regulates WRKY53 expression, and CAM5 and WRKY53 positively regulate SA-related gene expression. These proteins physically interact and form a complex that is unlocked by Ca2+ to release WRKY53. An electrophoretic mobility shift assay and dual-luciferase reporter assay demonstrated that WRKY53 and TGA5 cooperate to enhance the expression of the defense gene PATHOGENESIS-REALTED 1 (PR1) and that WRKY53 enhances the binding of TGA5 to the PR1 promoter. This paper proposes a framework that evk, as a RES substance, can achieve plants REScue through complete defense response.

plant biology↗

bioRxiv2022

Phenotypical Rescue of Bmp15 Deficiency by Mutation of Inhibin α (inha) Provides Novel Clues to How Bmp15 Controls Zebrafish Folliculogenesis

As an oocyte-specific growth factor, bone morphogenetic protein 15 (BMP15) plays a critical role in controlling folliculogenesis. However, the mechanism of BMP15 action remains elusive. Using zebrafish as the model, we created a bmp15 mutant using CRISPR/Cas9 and demonstrated that bmp15 deficiency caused a significant delay in follicle activation and puberty onset followed by complete arrest of follicle development at previtellogenic stage without yolk accumulation. The mutant females eventually underwent female-to-male sex reversal to become functional males, which was accompanied by a series of changes in secondary sexual characteristics. Interestingly, the blockade of folliculogenesis and sex reversal in bmp15 mutant could be rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic stage with yolk accumulation and the fish maintained their femaleness without sex reversal. Transcriptome analysis revealed up-regulation of pathways related to TGF-{beta} signaling and endocytosis in the double mutant follicles. Intriguingly, the expression of inhibin/activin {beta}Aa subunit (inhbaa) increased significantly in the double mutant ovary. Further knockout of inhbaa in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-) resulted in the loss of yolk granules again in the oocytes although the follicles could continue to grow beyond the size range of previtellogenic stage. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly in bmp15 single mutant females, returned to normal in the double mutant (bmp15-/-;inha-/-), but reduced again significantly in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-). E2 treatment could rescue the vitellogenic follicles in bmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation in bmp15-/-;inha-/- fish. In summary, the present study provided comprehensive genetic evidence for the interaction of bmp15 pathways and the activin-inhibin system in regulating folliculogenesis, in particular E2 production from the follicle, Vtg biosynthesis in the liver and its update by the developing oocytes.

developmental biology↗

bioRxiv2022

A sigmoid curve analysis method for pharmacological experimental results

Sigmoid curve (S-curve) is a basic exhibition form of dose-effect relationship in drug reaction. To analyze S-curve is an important method to well-understand drug reaction performance (DRP). The present study introduced an S-curve analysis method for pharmacological experiment results (PERs), the core of which was to solve the problem of the linear fitting of S-curve equation (S-Eq). The linear fitting Eqs of S-Eq were established with 100% fitness. Meanwhile, mathematical and pharmacological meaning of S-curve constants, ED50 and maximum effect (ymax) were clarified. The same group of experimental data was analyzed by the present method and four traditional analysis methods. The result indicates that the experimental parameters and their values displaying DRP got by different methods are different. The S-curve analysis method is closer to real drug reaction law.

pharmacology and toxicology↗

bioRxiv2022

Transcriptomic profiling reveals distinct subsets of immune checkpoint inhibitor-induced myositis

ObjectivesInflammatory myopathy or myositis is a heterogeneous family of immune-mediated diseases including dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM). Immune checkpoint inhibitors (ICI) can also cause myositis (ICI-myositis). This study was designed to define gene expression patterns in muscle biopsies from patients with ICI-myositis. MethodsBulk RNA sequencing was performed on 200 muscle biopsies (35 ICI-myositis, 44 DM, 18 AS, 54 IMNM, 16 IBM, and 33 normal muscle biopsies) and single nuclei RNA sequencing was performed on 22 muscle biopsies (7 ICI-myositis, 4 DM, 3 AS, 6 IMNM, and 2 IBM). ResultsUnsupervised clustering defined three distinct transcriptomic subsets of ICI-myositis: ICI-DM, ICI-MYO1, and ICI-MYO2. ICI-DM included patients with DM and anti-TIF1{gamma} autoantibodies who, like DM patients, overexpressed type 1 interferon-inducible genes. ICI-MYO1 patients had highly inflammatory muscle biopsies and included all patients that developed co-existing myocarditis. ICI-MYO2 was composed of patients with predominant necrotizing pathology and low levels of muscle inflammation. The type 2 interferon pathway was activated both in ICI-DM and ICI-MYO1. Unlike the other types of myositis, all three subsets of ICI-myositis patients overexpressed genes involved in the IL6 pathway. ConclusionsWe identified three distinct types of ICI-myositis based on transcriptomic analyses. The IL6 pathway was overexpressed in all groups, the type I interferon pathway activation was specific for ICI-DM, the type 2 IFN pathway was overexpressed in both ICIDM and ICI-MYO1, and only ICI-MYO1 patients developed myocarditis.

immunology↗

bioRxiv2022

Identifying Biomarkers of Retinal Pigment Epithelial Cell Stem Cell-derived RPE Cell Heterogeneity and Transplantation Efficacy

Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single cell RNA sequencing (sc-RNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed a lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.

cell biology↗

bioRxiv2022

Urine proteomic analysis of the rat e-cigarette model

Urinary proteomics was used to investigate the potential effects of e-cigarettes on the human body. In this study, a rat e-cigarette model was constructed by smoking for two weeks and urine samples before, during, and after e-cigarette smoking were collected. Urine proteomes before-after smoking of each rat were compared individually, while the control group was set up to rule out differences caused by rat growth and development. After smoking, the differential proteins produced by rats shows strong individual variation. Fetuin-B, a biomarker of COPD, and annexin A2, which is recognized as a multiple tumor marker, were identified as the differential proteins in five out of six smoking rats on day 3. To our surprise, odorant-binding proteins expressed in the olfactory epithelium were also found and were significantly upregulated, which may help explain olfactory adaptation. Pathways enriched by the differential proteins shows the evidence that smoking e-cigarettes affects the immune system, cardiovascular system, respiratory system, etc., which provides clues for further exploration of the mechanism of e-cigarettes on the human body.

biochemistry↗

bioRxiv2022

Arabidopsis CPK6 regulates drought tolerance under high nitrogen by the phosphorylation of NRT1.1

Nitrogen is an essential macronutrient for plant growth and development, and its availability to some extent is regulated by drought stress. Calcium-dependent protein kinases (CPKs) are a unique family of Ca2+ sensors with diverse functions in nitrogen and drought signaling pathways. However, which and how CPKs involve in the crosstalk between drought stress and nitrogen transportation remains largely unknown. Here we identified the drought tolerant function of Arabidopsis CPK6 under high nitrogen condition. The CPK6 expression is induced by the treatments of ABA and drought. The mutant cpk6 is insensitive to the ABA treatment, but sensitive to drought only under high nitrogen condition. CPK6 interacts with and phosphorylates the Thr571 in NRT1.1 protein, and thus represses its NO3- transporting activity under drought stress. Taken together, we showed the evidences that CPK6 regulates Arabidopsis drought tolerance through the phosphorylation of NRT1.1, and that enriches the knowledge of nitrogen uptake in plants during drought stress.

genetics↗

bioRxiv2022

Transient, developmental exposure to patient-derived anti-NMDA receptor autoantibodies causes long-term axonal and behavioral defects

NMDA receptor mediated autoimmune encephalitis (NMDAR-AE) frequently results in persistent sensory-motor deficits, especially in children, yet the underlying mechanisms remain unclear. This study investigated the long-term effects of exposure to a patient-derived GluN1-specific monoclonal antibody (mAb) during a critical developmental period (from postnatal day 3 to day 12) in mice. We observed long-lasting sensory-motor deficits characteristic of NMDAR-AE, along with permanent changes in callosal axons within the primary somatosensory cortex (S1) in adulthood, including increased terminal branch complexity. This complexity was associated with paroxysmal recruitment of neurons in S1 in response to callosal stimulation. Particularly during complex motor tasks, mAb3-treated mice exhibited significantly reduced inter-hemispheric functional connectivity between S1 regions, consistent with pronounced sensory-motor behavioral deficits. These findings suggest that transient exposure to anti-GluN1 mAb during a critical developmental window may lead to irreversible morphological and functional changes in callosal axons, which could significantly impair sensory-motor integration and contribute to long-lasting sensory-motor deficits. Our study establishes a new model of NMDAR-AE and identifies novel cellular and network-level mechanisms underlying persistent sensory-motor deficits in this context. These insights lay the foundation for future research into molecular mechanisms and the development of targeted therapeutic interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/510196v3_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@37f452org.highwire.dtl.DTLVardef@1894e46org.highwire.dtl.DTLVardef@171f954org.highwire.dtl.DTLVardef@1a81742_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Genetic Analysis of Activin/Inhibin β Subunits in Zebrafish Development and Reproduction

Activin and inhibin are both dimeric proteins sharing the same {beta} subunits that belong to the TGF-{beta} superfamily. They are well known for stimulating and inhibiting pituitary FSH secretion, respectively, in mammals. In addition, activin also acts as a mesoderm-inducing factor in frogs. However, their functions in development and reproduction of other species are poorly defined. In this study, we disrupted all three activin/inhibin {beta} subunits ({beta}Aa, inhbaa; {beta}Ab, inhbab; and {beta}B, inhbb) in zebrafish using CRISPR/Cas9. The loss of {beta}Aa/b but not {beta}B led to a high mortality rate in the post-hatching stage. Surprisingly, the expression of fshb but not lhb in the pituitary increased in the female {beta}A mutant together with aromatase (cyp19a1a) in the ovary. The single mutant of {beta}Aa/b showed normal folliculogenesis in young females; however, their double mutant (inhbaa-/-;inhbab-/-) showed delayed follicle activation, granulosa cell hypertrophy, stromal cell accumulation and tissue fibrosis. The ovary of inhbaa-/- deteriorated progressively after 180 dpf with reduced fecundity and the folliculogenesis ceased completely around 540 dpf. In addition, tumor- or cyst-like tissues started to appear in the inhbaa-/- ovary after about one year. In contrast to females, activin {beta}Aa/b mutant males showed normal spermatogenesis and fertility. As for activin {beta}B subunit, the inhbb-/- mutant exhibited normal folliculogenesis, spermatogenesis and fertility in both sexes; however, the fecundity of mutant females decreased dramatically at 270 dpf with accumulation of early follicles. In summary, the activin-inhibin system plays an indispensable role in fish reproduction, in particular folliculogenesis and ovarian homeostasis.

physiology↗

bioRxiv2022

Loss of H3.1K27me1 in Arabidopsis confers resistance to Geminivirus by sequestering DNA repair proteins onto rDNA and defense-related genes

The H3 methyltransferases ATXR5 and ATXR6 deposit H3.1K27me1 to heterochromatin to prevent genomic instability and transposon reactivation. Here, we report that atxr5 atxr6 mutants displayed robust resistance to Geminivirus. The viral resistance correlated with activation of DNA repair pathways, but not with transposon reactivation or heterochromatin amplification. We identified RAD51 and RPA1A as partners of virus-encoded Rep protein. The two DNA repair proteins showed increased binding to heterochromatic regions and defense-related genes in atxr5 atxr6 vs wild type plants. Consequently, the proteins had reduced interactions to viral DNA in the mutant, thus hampering viral replication. Additionally, RAD51 recruitment to the host genome arose via BRCA1, HOP2 and CYCB1, and this recruitment was essential for viral resistance in atxr5 atxr6. Thus, Geminiviruses adapt to healthy plants by hijacking its DNA repairing pathways for replication, but the host could retain DNA repairing proteins via sacrificing its genome stability to suppress viral infection.

genetics↗

bioRxiv2022

Tracking neural correlates of prioritizing working memory representations through retrospective attentional strengthening

Previous work has proposed two potentials benefits of retrospective attention on working memory (WM): target strengthening and non-target inhibition. It remains unknown which hypothesis contributes to the improved WM performance, yet the neural mechanisms responsible for this attentional benefit are unclear. Here, we recorded electroencephalography (EEG) signals while 33 participants performed a retrospective-cue WM task. Multivariate pattern classification analysis revealed that only representations of target features were enhanced by valid retrospective attention during the retention, supporting the target strengthening hypothesis. Further univariate analysis found that mid-frontal theta inter-trial phase coherence (ITPC) and ERP components were modulated by valid retrospective attention and correlated with individual differences and moment-to-moment fluctuations on behavioral outcomes, suggesting that both trait- and state-level variability in attentional preparatory processes influence goal-directed behavior. Furthermore, task-irrelevant target spatial location could be decoded from EEG signals, indicating that enhanced spatial binding of target representation promotes high WM precision. Importantly, frontoparietal theta-alpha phase-amplitude-coupling was increased by valid retrospective attention and predicted the reduced randomly guessing rates. This long-range connection supported top-down information flow in engagement of frontoparietal networks, which might organize attentional states to integrate target features. Altogether, these results provide neurophysiological bases that retrospective attention improves WM precision through enhancing representation of target and emphasize the critical role of frontoparietal attentional network in the control of WM representations.

neuroscience↗

bioRxiv2022

Single-cell Atlas of Human Fetal Cerebellum Decoding Medulloblastoma Origin and Oncogenesis

Medulloblastoma (MB) is the most common malignant childhood brain tumor1,2, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella3,4 or human counterparts from frozen tissue nuclei5 have not fully defined the compositional heterogeneity of MBs. Here, we undertook an unprecedented single-cell profiling of freshly-isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group-3 and metastatic tumors. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group-3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin-loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group-3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group-3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs reveal potential cell populations predisposed to transformation and regulatory circuitries underlying tumor cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

cancer biology↗

bioRxiv2022

Transformer Enables Reference Free And Unsupervised Analysis of Spatial Transcriptomics

The development of spatial transcriptomics technologies makes it possible to study tissue heterogeneity at the scale of spatial expressed microenvironment. However, most of the previous methods collapse the spatial patterns in the low spatial resolution. Existing reference based deconvolution methods integrate single-cell reference and spatial transcriptomics data to predict the proportion of cell-types, but the availability of suitable single-cell reference is often limited. In this paper, we propose a novel Transformer based model (TransfromerST) to integrate the spatial gene expression measurements and their spatial patterns in the histology image (if available) without single cell reference. TransfromerST enables the learning of the locally realistic and globally consistent constituents at nearly single cell resolution. TransfromerST firstly uses a transformer based variational autoencoder to explore the latent representation of gene expression, which is further embedded with the spatial relationship learned from adaptive graph Transformer model. The super-resolved cross-scale graph network improves the model-fit to enhanced structure-functional interactions. The public and in-house experimental results with multimodal spatial transcriptomics data demonstrate TransfromerST could highlight the tissue structures at nearly single cell resolution and detect the spatial variable genes and meta gene for each spatial domain. In summary, TransfromerST provides an effective and efficient alternative for spatial transcriptomics tissue clustering, super-resolution and gene expression prediction from histology image.

bioinformatics↗

bioRxiv2022

Neutralization of Omicron BA.4/BA.5 and BA.2.75 by Booster Vaccination or BA.2 Breakthrough Infection Sera

Many new Omicron sub-lineages have been reported to evade neutralizing antibody response, including BA.2, BA.2.12.1, BA.4 and BA.5. Most recently, another emerging sub-lineage BA.2.75 has been reported in multiple countries. In this study, we constructed a comprehensive panel of pseudoviruses (PsVs), including wild-type, Delta, BA.1, BA.1.1, BA.2, BA.3, BA.2.3.1, BA.2.10.1, BA.2.12.1, BA.2.13, BA.2.75 and BA.4/BA.5, with accumulate coverage reached 91% according to the proportion of sequences deposited in GISAID database since Jan 1st, 2022. We collected serum samples from healthy adults at day14 post homologous booster with BBIBP-CorV, or heterologous booster with ZF2001, primed with two doses of BBIBP-CorV, or from convalescents immunized with three-dose inactivated vaccines prior to infection with Omicron BA.2, and tested their neutralization activity on this panel of PsVs. Our results demonstrated that all Omicron sub-lineages showed substantial evasion of neutralizing antibodies induced by vaccination and infection, although BA.2.75 accumulated the largest number of mutations in its spike, BA.4 and BA.5 showed the strongest serum escape. However, BA.2 breakthrough infection could remarkably elevated neutralization titers against all different variants, especially titers against BA.2 and its derivative sub-lineages.

immunology↗

bioRxiv2022

ModelArray: a memory-efficient R package for statistical analysis of fixel data

Diffusion MRI is the dominant non-invasive imaging method used to characterize white matter organization in health and disease. Increasingly, fiber-specific properties within a voxel are analyzed using fixels. While tools for conducting statistical analyses of fixel data exist, currently available tools are memory intensive, difficult to scale to large datasets, and support only a limited number of statistical models. Here we introduce ModelArray, a memory-efficient R package for mass-univariate statistical analysis of fixel data. With only several lines of code, even large fixel datasets can be analyzed using a standard personal computer. At present, ModelArray supports linear models as well as generalized additive models (GAMs), which are particularly useful for studying nonlinear effects in lifespan data. Detailed memory profiling revealed that ModelArray required only limited memory even for large datasets. As an example, we applied ModelArray to fixel data derived from diffusion images acquired as part of the Philadelphia Neurodevelopmental Cohort (n=938). ModelArray required far less memory than existing tools and revealed anticipated nonlinear developmental effects in white matter. Moving forward, ModelArray is supported by an open-source software development model that can incorporate additional statistical models and other imaging data types. Taken together, ModelArray provides an efficient and flexible platform for statistical analysis of fixel data. HIGHLIGHTSO_LIModelArray is an R package for mass-univariate statistical analysis of fixel data C_LIO_LIModelArray is memory-efficient even for large-scale datasets C_LIO_LIModelArray supports linear and nonlinear modeling and is extensible to more models C_LIO_LIModelArray facilitates easy statistical analysis of large-scale fixel data C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/499631v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1946469org.highwire.dtl.DTLVardef@14c3076org.highwire.dtl.DTLVardef@10197beorg.highwire.dtl.DTLVardef@19170cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Pitfalls of genotyping microbial communities with rapidly growing genome collections

Detecting genetic variants in metagenomic data is a priority for understanding the evolution, ecology, and functional characteristics of microbial communities. Many recent tools that perform this metagenotyping rely on aligning reads of unknown origin to a reference database of sequences from many species before calling variants. Using simulations designed to represent a wide range of scenarios, we demonstrate that diverse and closely related species both reduce the power and accuracy of reference-based metagenotyping. We identify multi-mapping reads as a prevalent source of errors and illustrate a tradeoff between retaining correct alignments versus limiting incorrect alignments, many of which map reads to the wrong species. Then we quantitatively evaluate several actionable mitigation strategies and review emerging methods with promise to further improve metagenotyping. These findings document a critical challenge that has come to light through the rapid growth of genome collections that push the limits of current alignment algorithms. Our results have implications beyond metagenotyping to the many tools in microbial genomics that depend upon accurate read mapping. HIGHLIGHTSO_LIMost microbial species are genetically diverse. Their single nucleotide variants can be genotyped using metagenomic data aligned to databases constructed from genome collections ("metagenotyping"). C_LIO_LIMicrobial genome collections have grown and now contain many pairs of closely related species. C_LIO_LIClosely related species produce high-scoring but incorrect alignments while also reducing the uniqueness of correct alignments. Both cause metagenotype errors. C_LIO_LIThis dilemma can be mitigated by leveraging paired-end reads, customizing databases to species detected in the sample, and adjusting post-alignment filters. C_LI

bioinformatics↗

bioRxiv2022

MIDAS2: Metagenomic Intra-species Diversity Analysis System

SummaryThe Metagenomic Intra-Species Diversity Analysis System (MIDAS) is a scalable metagenomic pipeline that identifies single nucleotide variants (SNVs) and gene copy number variants (CNVs) in microbial populations. Here, we present MIDAS2, which addresses the computational challenges presented by increasingly large reference genome databases, while adding functionality for building custom databases and leveraging paired-end reads to improve SNV accuracy. This fast and scalable reengineering of the MIDAS pipeline enables thousands of metagenomic samples to be efficiently genotyped. Availability and ImplementationThe source code is available at https://github.com/czbiohub/MIDAS2. The documentation is available at https://midas2.readthedocs.io/en/latest/. Supplementary InformationSupplementary data are available at Bioinformatics online.

bioinformatics↗