Biology subjects

Shi, L.

Publications and source records attributed to Shi, L..

At least 163 records · Page 9Linked to original sources

bioRxiv2023

Imaging Architecture of Granulomas Induced by Mycobacterium tuberculosis Infections with Single-Molecule FISH

Granulomas are an important hallmark of Mycobacterium tuberculosis (Mtb) infection. They are organized and dynamic structures created by an assembly of immune cells around the sites of infection in the lungs to locally restrict the bacterial growth and the hosts inflammatory responses. The cellular architecture of granulomas is traditionally studied by immunofluorescence labeling of phenotypic surface markers. However, very few antibodies are available for model animals used in tuberculosis research, such as non-human primates and rabbits; secreted immunological markers such as cytokines cannot be imaged in situ using antibodies; and traditional phenotypic surface markers do not provide sufficient resolution for the detection of many subtypes and differentiation states of immune cells. Using single-molecule fluorescent in situ hybridization (smFISH) and its derivatives, amplified smFISH (ampFISH) and iterative smFISH, we developed a platform for imaging mRNAs encoding immune markers in rabbit and macaque tuberculosis granulomas. Multiplexed imaging for several mRNA and protein markers was followed by quantitative measurement of expression of these markers in single cells in situ. A quantitative analysis of combinatorial expressions of these markers allowed us to classify the cells into several subtypes and chart their distributions within granulomas. For one mRNA target, HIF-1, we were able to image its mRNA and protein in the same cells, demonstrating the specificity of probes. This method paves the way for defining granular differentiation states and cell subtypes from transcriptomic data, identifying key mRNA markers for these cell subtypes, and then locating the cells in the spatial context of granulomas.

immunology↗

bioRxiv2023

Reprogramming Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD1 Therapy

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Anti-PD-L1 immunotherapy combined with gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers, but responses are seen only in a minority of patients. Here, we studied the roles of anti-PD1 and anti-CTLA-4 immune checkpoint blockade (ICB) therapies when combined with gemcitabine/cisplatin and the mechanisms of treatment benefit in orthotopic murine ICC models. We evaluated the effects of the combined treatments on ICC vasculature and immune microenvironment using flow cytometry analysis, immunofluorescence, imaging mass cytometry, RNA-sequencing, qPCR, and in vivo T-cell depletion and CD8+ T-cell transfer using orthotopic ICC models and transgenic mice. Combining gemcitabine/cisplatin with anti-PD1 and anti-CTLA-4 antibodies led to substantial survival benefits and reduction of morbidity in two aggressive ICC models, which were ICB-resistant. Gemcitabine/cisplatin treatment increased the frequency of tumor-infiltrating lymphocytes and normalized the ICC vessels, and when combined with dual CTLA-4/PD1 blockade, increased the number of activated CD8+Cxcr3+IFN-{gamma}+ T-cells. Depletion of CD8+ but not CD4+ T-cells compromised efficacy. Conversely, CD8+ T-cell transfer from Cxcr3-/- versus Cxcr3+/+ mice into Rag1-/- immunodeficient mice restored the anti-tumor effect of gemcitabine/cisplatin/ICB combination therapy. Finally, rational scheduling of the ICBs (anti-CTLA-4 "priming") with chemotherapy and anti-PD1 therapy achieved equivalent efficacy with continuous dosing while reducing overall drug exposure. In summary, gemcitabine/cisplatin chemotherapy normalizes vessel structure, increases activated T-cell infiltration, and enhances anti-PD1/CTLA-4 immunotherapy efficacy in aggressive murine ICC. This combination approach should be clinically tested to overcome resistance to current therapies in ICC patients. One Sentence SummaryImmune microenvironment reprogramming by chemotherapy and priming using CTLA-4 blockade render ICCs responsive to anti-PD-1 immunotherapy.

cancer biology↗

bioRxiv2023

A cinnamyl alcohol dehydrogenase (CAD) like enzyme leads to a branch in the shikonin biosynthetic pathway in Arnebia euchroma

Shikonin derivatives are natural naphthoquinone compounds and the main bioactive components produced by several boraginaceous plants, such as Lithospermum erythrorhizon and Arnebia euchroma. Phytochemical researches utilizing L. erythrorhizon and A. euchroma cultured cells both indicate the existence of a competing route branching out from the shikonin biosynthetic pathway toward benzo/hydroquinones. A previous study has shown that the branch point is a putative alcohol dehydrogenase converting (Z)-3-hydroxygeranylhydroquinone [(Z)-3-OH-GHQ] to an aldehyde intermediate (E)-3-oxo-GHQ. However, the enzyme involved in the branch reaction is not characterized at the molecular level yet. In this study, we clone a candidate gene belonging to the cinnamyl alcohol dehydrogenase (CAD) family, AeHGO, through coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines of A. euchroma. In biochemical assays, purified AeHGO protein reversibly oxidizes (Z)-3-OH-GHQ to produce (E)-3-oxo-GHQ followed by reversibly reducing (E)-3-oxo-GHQ to (E)-3-OH-GHQ, resulting in an equilibrium mixture of the three compounds. Time course analysis and kinetic parameters show that the reaction with (Z)-3-OH-GHQ is about twice as efficient as with (E)-3-OH-GHQ, which leads to the predominance of (E)-3-OH-GHQ and (E)-3-oxo-GHQ in the equilibrium mixture. According to a previous report, (E)-3-oxo-GHQ can be converted to deoxyshikonofuran, a hydroquinone metabolite produced by boraginaceous plants. Considering there is a competition for accumulation between shikonin derivatives and benzo/hydroquinones in both L. erythrorhizon and A. euchroma cultured cells, AeHGO is supposed to play an important role in the metabolic regulation of shikonin biosynthetic pathway.

biochemistry↗

bioRxiv2022

Quantitative measures of topographic and divergent/convergent connectivity in diffusion MRI of the human cerebral cortex

Spatial features of connections, such as topography and divergence/convergence, reflect the information-processing mechanisms crucial for understanding and modeling the brain. However, this has not yet been comprehensively investigated. Using diffusion Magnetic Resonance Imaging (dMRI) data, we developed a topographic factor (TF) and divergence/convergence factor (DC) to quantitatively explore the spatial connectivity patterns of one entire hemisphere of the human cerebral cortex. In the analysis, the early sensory areas, which are located far away from the center of the cerebral cortex, exhibited high topographic connectivity. In contrast, the limbic system, which is located proximal to the center, showed high divergence/convergence in two types of connectivity: one region to another region at the region-to-region level, and one region to all other regions at the global level. Topography had anti-correlation with divergence/convergence over the cerebral cortex, and the two types of divergence/convergence displayed positive correlation with each other. These results suggest that topographic and divergent/convergent connectivity are spatially organized with respect to cytoarchitecture over the cerebral cortex to optimize energy efficiency and information transfer performance in the cerebral cortex.

neuroscience↗

bioRxiv2022

Modulating the epigenetic state promotes the reprogramming of transformed cells to pluripotency in a line-specific manner

Somatic cell reprogramming and oncogenic transformation share surprisingly similar features, yet transformed cells are highly resistant to reprogramming. There must be barriers that block transformed cells from reprogramming, but the nature of those barriers is unclear. In this study, we generated a systematic panel of transformed mouse embryonic fibroblasts (MEFs) using a variety of oncogenic transgenes, and discovered transformed cell lines that remain compatible with reprogramming when transfected with Oct4/Sox2/Klf4/Myc. By comparing the reprogramming-capable and incapable transformed lines we identified multiple stages of failure in the reprogramming process. Some transformed lines failed very early, whilst other lines seemed to progress through a normal-looking reprogramming process. Finally, we show that MEK inhibition overcomes one critical reprogramming barrier by indirectly suppressing a hyperactive epigenetic state in some of the transformed cells. This study reveals that the barriers underlying resistance to reprogramming vary between the different transformation methods. Key findingsO_LISomatic cell reprogramming of transformed cells is context-specific C_LIO_LIInhibition of MEK converts some cell lines to reprogramming-capable C_LIO_LITransformed cell lines are characterized by a hyperactive chromatin state C_LIO_LIMEK inhibition indirectly affects chromatin to enable reprogramming C_LI

cell biology↗

bioRxiv2022

BDNF and TRiC-inspired Reagents Rescue Cortical Synaptic Deficits in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) results from a CAG repeat expansion in the gene for Huntington (HTT) resulting in expansion of the polyglutamine (Q) tract in the mutant protein (mHTT). Synaptic changes are early manifestations of neuronal dysfunction in HD. However, the mechanism(s) by which mHTT impacts synapse formation and function is not well defined. Herein we explored HD pathogenesis in the BACHD and the {Delta}N17-BACHD mouse models of HD by examining cortical synapse formation and function in primary cultures maintained for up to 35 days (DIV35). We identified synapses by immunostaining with antibodies against pre-synaptic (Synapsin 1) and a post-synaptic (PSD95) marker. Consistent with earlier studies, cortical neurons from both WT and the HD models began to form synapses at DIV14; at this age there were no genotypic differences in synapse numbers. However, from DIV21 through DIV35 BACHD neurons showed progressively smaller numbers of synapses relative to WT neurons. Remarkably, BACHD synaptic deficits were completely rescued by treating cultures with BDNF. Building on earlier studies using reagents inspired by the chaperonin TRiC, we found that addition of the recombinant apical domain of CCT1 partially rescued synapse number. Unexpectedly, unlike BACHD cultures, synapses in {Delta}N17-BACHD cultures showed a progressive increase in number as compared to WT neurons, thus distinguishing synaptic changes in these HD models. Using multielectrode arrays, we discovered age-related functional deficits in BACHD cortical cultures with significant differences present by DIV28. As for synapse number, BDNF treatment prevented most synaptic deficits, including mean firing rate, spikes per burst, inter-burst interval, and synchrony. The apical domain of CCT1 showed similar, albeit less potent effects. These data are evidence that deficits in HD synapse number and function can be replicated in vitro and that treatment with either BDNF or a TRiC-inspired reagent can prevent them. Our findings support the use of cellular models to further explicate HD pathogenesis and its treatments.

neuroscience↗

bioRxiv2022

Starfysh reveals heterogeneous spatial dynamics in the breast tumor microenvironment

Spatially-resolved gene expression profiling provides valuable insight into tissue organization and cell-cell crosstalk; however, spatial transcriptomics (ST) lacks single-cell resolution. Current ST analysis methods require single-cell RNA sequencing data as a reference for a rigorous interpretation of cell states and do not utilize associated histology images. Significant sample variation further complicates the integration of ST datasets, which is essential for identifying commonalities across tissues or altered cellular wiring in disease. Here, we present Starfysh, the first comprehensive computational toolbox for joint modeling of ST and histology data, dissection of refined cell states, and systematic integration of multiple ST datasets from complex tissues. Starfysh uses an auxiliary deep generative model that incorporates archetypal analysis and any known cell state markers to avoid the need for a single-cell-resolution reference in characterizing known or novel tissue-specific cell states. Additionally, Starfysh improves the characterization of spatial dynamics in complex tissues by leveraging histology images and enables the comparison of niches as spatial "hubs" across tissues. Integrative analysis of primary estrogen receptor-positive (ER+) breast cancer, triple-negative breast cancer (TNBC), and metaplastic breast cancer (MBC) tumors using Starfysh led to the identification of heterogeneous patient- and disease-specific hubs as well as a shared stromal hub with varying spatial orientation. Our results show the ability to delineate the spatial co-evolution of tumor and immune cell states and their crosstalk underlying intratumoral heterogeneity in TNBC and revealed metabolic reprogramming shaping immunosuppressive hubs in aggressive MBC. Starfysh is publicly available (https://github.com/azizilab/starfysh).

bioinformatics↗

bioRxiv2022

Identification and Characterization of ML321: a Novel and Highly Selective D2 Dopamine Receptor Antagonist with Efficacy in Animal Models that Predict Atypical Antipsychotic Activity

We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity - ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit "atypical" antipsychotic activity in humans with significantly fewer side effects than observed with currently FDA-approved D2R antagonists.

pharmacology and toxicology↗

bioRxiv2022

Quartet metabolite reference materials for assessing inter-laboratory reliability and data integration of metabolomic profiling

Various laboratory-developed metabolomic methods lead to big challenges in inter-laboratory comparability and effective integration of diverse datasets. As part of the Quartet Project, we established a publicly available suite of four metabolite reference materials derived from B-lymphoblastoid cell lines from a family quartet of parents and monozygotic twin daughters. We generated comprehensive LC-MS based metabolomic data from the Quartet reference materials using targeted and untargeted strategies in different laboratories. High variabilities in the qualitative and quantitative metabolomic measurements were observed across laboratories. Moreover, the Quartet multi-sample-based quality metrics were developed for objectively assessing the reliability of metabolomic profiling in detecting intrinsic biological differences among difference groups of samples. Importantly, the ratio-based metabolomic profiling, by scaling the absolute values of a study sample relative to those of a universal reference sample, enables data integration in long-term measurements across difference laboratories or platforms. Thus, we constructed the ratio-based high-confidence reference datasets between two reference samples, providing "ground truth" for inter-laboratory proficiency test, which enables objective assessment of various metabolomic methods. Our study provided the community with rich resources and best practice for objective assessment of inter-laboratory measurements and data integration, ensuring reliable large-scale and longitudinal metabolomic profiling.

systems biology↗

bioRxiv2022

Probing lung function at high spatiotemporal resolution using a novel crystal ribcage

Understanding the dynamic pathogenesis and treatment response in pulmonary diseases requires probing the lung at cellular resolution in real-time. Despite recent progress in intravital imaging, optical imaging of the lung during active respiration and circulation has remained challenging. Here, we introduce the crystal ribcage: a transparent ribcage that (i) allows truly multiscale optical imaging of the lung in health and disease from whole-organ to single cell, (ii) enables the modulation of lung biophysics and immunity through intravascular, intrapulmonary, intraparenchymal, and optogenetic interventions, and (iii) preserves the 3-D architecture, air-liquid interface, cellular diversity, and respiratory-circulatory functions of the lung. Utilizing these unprecedented capabilities on murine models of primary and metastatic lung tumors, respiratory infection, pulmonary fibrosis, emphysema, and acute lung injury we probed how disease progression remodels the respiratory-circulatory functions at the single alveolus and capillary levels. In cancer, we identified the earliest stage of tumorigenesis that compromises alveolar and capillary functions, a key state with consequences on tumor progression and treatment response. In pneumonia, we mapped mutual links between the recruited immune cells and the alveolar-capillary functions. We found that neutrophil migration is strongly and reversibly responsive to vascular pressure with implications for understanding of how lung physiology, altered by disease and anatomical location, affects immune cell activities. The crystal ribcage and its broad applications presented here will facilitate further studies of real-time remodeling of the alveoli and capillaries during pathogenesis of nearly any pulmonary disease, leading to the identification of new targets for treatment strategies.

bioengineering↗

bioRxiv2022

Ratio-based quantitative multiomics profiling using universal reference materials empowers data integration

Multiomics profiling is a powerful tool to characterize the same samples with complementary features orchestrating the genome, epigenome, transcriptome, proteome, and metabolome. However, the lack of ground truth hampers the objective assessment of and subsequent choice from a plethora of measurement and computational methods aiming to integrate diverse and often enigmatically incomparable omics datasets. Here we establish and characterize the first suites of publicly available multiomics reference materials of matched DNA, RNA, proteins, and metabolites derived from immortalized cell lines from a family quartet of parents and monozygotic twin daughters, providing built-in truth defined by family relationship and the central dogma. We demonstrate that the "ratio"-based omics profiling data, i.e., by scaling the absolute feature values of a study sample relative to those of a concurrently measured universal reference sample, were inherently much more reproducible and comparable across batches, labs, platforms, and omics types, thus empower the horizontal (within-omics) and vertical (cross-omics) data integration in multiomics studies. Our study identifies "absolute" feature quantitation as the root cause of irreproducibility in multiomics measurement and data integration, and urges a paradigm shift from "absolute" to "ratio"-based multiomics profiling with universal reference materials.

systems biology↗

bioRxiv2022

Correcting batch effects in large-scale multiomic studies using a reference-material-based ratio method

Batch effects are notorious technical variations that are common in multiomic data and may result in misleading outcomes. With the era of big data, tackling batch effects in multiomic integration is urgently needed. As part of the Quartet Project for quality control and data integration of multiomic profiling, we comprehensively assess the performances of seven batch-effect correction algorithms (BECAs) for mitigating the negative impact of batch effects in multiomic datasets, including transcriptomics, proteomics, and metabolomics. Performances are evaluated based on accuracy of identifying differentially expressed features, robustness of predictive models, and the ability of accurately clustering cross-batch samples into their biological sample groups. Ratio-based method is more effective and widely applicable than others, especially in cases when batch effects are highly confounded with biological factors of interests. We further provide practical guidelines for the implementation of ratio-based method using universal reference materials profiled with study samples. Our findings show the promise for eliminating batch effects and enhancing data integration in increasingly large-scale, cross-batch multiomic studies.

systems biology↗

bioRxiv2022

FGFR1 governs iron metabolism via regulating post-translational modification of IRP2 in prostate cancer cells

The acquisition of ectopic fibroblast growth factor receptor 1 (FGFR1) expression is well documented in prostate cancer (PCa) progression. However, how FGFR1 facilitates PCa progression is not fully revealed, although it is known to confer tumor growth advantage and metastasis. Here we report that FGFR1 deletion in DU145 human PCa cells retards the iron metabolism and reduces transferrin receptor 1 (TFR1), which synergistically enhances the anti-cancer effect of iron chelator. FGFR1 and TFR1 are highly expressed in PCa, and FGFR1 overexpression increased TFR1 in PCa cell lines. Furthermore, we first time demonstrate that FGFR1 deletion boosts and shifts the degradation of iron regulatory proteins 2 (IRP2) to downregulate TFR1. Detailed characterization revealed that based on FGFR1 deletion the stability of IRP2 is broken, whose degradation is accelerated, which can be not observed without FGFR1 deletion. In addition, IRP2 overexpression rescue the malignancy degree of DU145 cells. Our results here unravel a novel mechanism by which FGFR1 promotes PCa progression by upregulating iron metabolism, and that the FGFR1/IRP2/TFR1 axis can be a potential target for managing PCa progression.

cancer biology↗

bioRxiv2022

Chromatin regulators in the TBX1 network confer risk for conotruncal heart defects in 22q11.2DS and sporadic congenital heart disease

BackgroundCongenital heart disease (CHD) affecting the conotruncal region of the heart, occur in half of patients with 22q11.2 deletion syndrome. This syndrome is a rare disorder with relative genetic homogeneity that can facilitate identification of genetic modifiers. Haploinsufficiency of TBX1, mapped to the 22q11.2 region, encoding a T-box transcription factor, is one of the main genes for the etiology of the syndrome. We suggest that genetic modifiers of CHD in patients with 22q11.2 deletion syndrome may be in the TBX1 gene network. MethodsTo identify genetic modifiers of 22q11.2 deletion syndrome, we analyzed whole genome sequence of subjects with 22q11.2DS, of which 456 were cases with conotruncal heart defects and 537 were controls with normal cardiac structures. We retained the most damaging rare coding variants and examined 19 functional gene sets for association that were weighted upon expression of genes in cardiac progenitor cells in mouse embryos identified by RNA-sequencing. ResultsWe identified rare damaging coding variants in chromatin regulatory genes as modifiers of conotruncal heart defects in 22q11.2DS. Chromatin genes with recurrent damaging variants include EP400, KAT6A, KMT2C, KMT2D, NSD1, CHD7 and PHF21A. In total, we identified 37 chromatin regulatory genes, that may increase risk for conotruncal heart defects in 8.5% of 22q11.2 deletion syndrome cases. Many of these genes were identified as risk factors for sporadic CHD in the general population increasing the likelihood that these genes are medically important contributors for CHD. These genes are co-expressed in cardiac progenitor cells with TBX1, suggesting that they may be in the same genetic network. Some of the genes identified, such as KAT6A, KMT2C, CHD7 and EZH2, have been previously shown to genetically interact with TBX1 in mouse models, providing mechanistic validation of these genes found. ConclusionsOur findings indicate that disturbance of chromatin regulatory genes impact a TBX1 gene network serving as genetic modifiers of 22q11.2 deletion syndrome. Since some of these chromatin regulatory genes were found in individuals with sporadic CHD, we suggest that there are shared mechanisms involving the TBX1 gene network in the etiology of CHD.

genetics↗

bioRxiv2022

Optical Metabolic Imaging Uncovers Sex- and Diet-dependent Lipid Changes in Aging Drosophila Brain

Aging is associated with progressive declines in physiological integrity and functions alongside increases in vulnerability to develop a number of diseases. The brain regulates sensory and motor functions as well as endocrine functions, and age-associated changes in brain are likely prerequisite for the organismal aging. Lipid metabolism has been associated with brain aging, which could be easily intervened by diets and lifestyles. However, the underlying mechanism through which brain lipid metabolism is regulated by diet during aging is elusive. Using stimulated Raman scattering (SRS) imaging combined with deuterium water (D2O) labeling, we visualized that lipid metabolic activities were changed by diet manipulation in aging Drosophila brain. Furthermore, we illuminated that insulin/IGF-1 signaling (IIS) pathway mediates the transformation of brain lipid metabolic changes in both an aging- and a diet-dependent manner. The lipid droplets (LDs) in the brain gradually became inert in both activities of lipid synthesis and mobilization with aging. High sugar diets enhanced the metabolic activity through promoting lipogenesis while dietary restriction increased the metabolic activity in both lipogenesis and lipolysis in brain LDs. However, these effects were impaired in both chico1/+ and dfoxo Drosophila mutants. We also observed that old chico1/+ brains maintained high metabolic activities, whilst the aged dfoxo brains acted exactly the opposite. More interestingly, the sexual dimorphism in brain lipid metabolism was impaired under diet regulation in both chico1/+ and dfoxo mutants. Locally reduced IIS activity in glial cells can mimic the systemic changes in systematic IIS mutants to maintain lipogenesis and lipolysis in aged brains, providing mechanistic insight into the anti-aging effects of IIS pathway. Our results highlight the manipulation of glia-specific IIS activity as a promising strategy in anti-aging treatments.

bioengineering↗

bioRxiv2022

The Quartet Data Portal: integration of community-wide resources for multiomics quality control

The implementation of quality control for multiomic data requires the widespread use of well-characterized reference materials, reference datasets, and related resources. The Quartet Data Portal was built to facilitate community access to such rich resources established in the Quartet Project. A convenient platform is provided for users to request the DNA, RNA, protein, and metabolite reference materials, as well as multi-level datasets generated across omics, platforms, labs, protocols, and batches. Interactive visualization tools are offered to assist users to gain a quick understanding of the reference datasets. Crucially, the Quartet Data Portal continuously collects, evaluates, and integrates the community-generated data of the distributed Quartet multiomic reference materials. In addition, the portal provides analysis pipelines to assess the quality of user-submitted multiomic data. Furthermore, the reference datasets, performance metrics, and analysis pipelines will be improved through periodic review and integration of multiomic data submitted by the community. Effective integration of the evolving technologies via active interactions with the community will help ensure the reliability of multiomics-based biological discoveries. The Quartet Data Portal is accessible at https://chinese-quartet.org. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

systems biology↗

bioRxiv2022

Quartet DNA reference materials and datasets for comprehensively evaluating germline variants calling performance

Current methods for evaluating the accuracy of germline variant calls are restricted to easy-to-detect high-confidence regions, thus ignoring a substantial portion of difficult variants beyond the benchmark regions. We established four DNA reference materials from immortalized cell lines derived from a Chinese Quartet including parents and monozygotic twins. We integrated benchmark calls of 4.2 million small variants and 15,000 structural variants from multiple platforms and bioinformatic pipelines for evaluating the reliability of germline variant calls inside the benchmark regions. The genetic built-in-truth of the Quartet family design not only improved sensitivity of benchmark calls by removing additional false positive variants with apparently high quality, but also enabled estimation of the precision of variants calls outside the benchmark regions. Batch effects of variant calling in large-scale DNA sequencing efforts can be effectively identified with the concurrent use of the Quartet DNA reference materials along with study samples, and can be alleviated by training a machine learning model with the Quartet reference datasets to remove potential artifact calls. Matched RNA and protein reference materials were also established in the Quartet project, thereby enabling benchmark calls constructed from DNA reference materials for evaluation of variants calling performance on RNA and protein data. The Quartet DNA reference materials from this study are a resource for objective and comprehensive assessment of the accuracy of germline variant calls throughout the whole-genome regions.

bioinformatics↗

bioRxiv2022

Quartet RNA reference materials and ratio-based reference datasets for reliable transcriptomic profiling

As an indispensable tool for transcriptome-wide analysis of differential gene expression, RNA sequencing (RNAseq) has demonstrated great potential in clinical applications. However, the lack of multi-group RNA reference materials of biological relevance and the corresponding reference datasets for assessing the reliability of RNAseq hampers its wide clinical applications wherein the underlying biological differences among study groups are often small. As part of the Quartet Project for quality control and data integration of multiomic profiling, we established four RNA reference materials derived from immortalized B-lymphoblastoid cell lines from four members of a monozygotic twin family. Additionally, we constructed ratio-based transcriptome-wide reference datasets using multi-batch RNAseq datasets, providing "ground truth" for benchmarking. Moreover, Quartet-sample-based quality metrics were developed for assessing reliability of RNAseq technology in terms of intra-batch proficiency and cross-batch reproducibility. The small intrinsic biological differences among the Quartet samples enable sensitive assessment of performance of transcriptomic measurements. The Quartet RNA reference materials combined with the reference datasets can be served as unique resources for assessing data quality and improving reliability of transcriptomic profiling.

genetics↗