Biology subjects

Zhou, S.

Publications and source records attributed to Zhou, S..

At least 145 records · Page 8Linked to original sources

bioRxiv2023

The roles of DDR2 and substrate stiffness on cancer cell transcriptome and proliferation

The interactions between cancer cells and the ECM regulate carcinogenesis. The collagen receptor kinase DDR2 is dysregulated in certain cancer cells, but its precise role in these malignancies remains unclear. In this study, we perform RNA-seq to determine how DDR2 and the biomechanical environment regulate cancer cell behaviors. We show that DDR2 knockdown in SH-SY5Y neuroblastoma cells inhibits proliferation and promotes senescence by regulating relevant genes. Increasing substrate stiffness reduces proliferation and promotes cell spreading but does not change senescence or transcriptome. Furthermore, DDR2 knockdown modulates cellular responses to substrate stiffness changes, unraveling a crosstalk between DDR2 and mechanosensing. These findings indicate DDR2 and ECM biomechanics regulate cancer cell behavior through distinct mechanisms, providing new mechanistic insights of cancer progression.

biochemistry↗

bioRxiv2023

Integrative analysis of the methylome and transcriptome of tomato fruit (Solanum lycopersicum L.) induced by postharvest handling

Tomato fruit ripening is triggered by the demethylation of key genes, which alters their transcriptional levels thereby initiating and propagating a cascade of physiological events. What is unknown, is how these processes are altered when fruit are ripened using postharvest practices to extend shelf-life, as these practices often reduce fruit quality. To address this, postharvest handling-induced changes in the fruit DNA methylome and transcriptome, and how they correlated with ripening speed, and ripening indicators such as ethylene, ABA, and carotenoids, were assessed. This study comprehensively connected changes in physiological events with dynamic molecular changes. Ripening fruit that reached Turning (T) after storage under dark at 20{degrees}C, 12.5{degrees}C, or 5{degrees}C chilling (followed by 20{degrees}C rewarming), were compared to fresh-harvest fruit FHT. Fruit stored at 12.5{degrees}C, had the biggest epigenetic marks and alterations in gene expression, exceeding changes induced by postharvest chilling. Fruit physiological and chronological age were uncoupled at 12.5{degrees}C, as the time-to-ripening was longest. Fruit ripening at 12.5{degrees}C was not climacteric; there was no respiratory or ethylene burst, rather, fruit were high in ABA. Clear differentiation between postharvest-ripened and FHT was evident in the methylome and transcriptome. Higher expression of photosynthetic genes and chlorophyll levels in FHT fruit, pointing to light as influencing the molecular changes in fruit ripening. Finally, correlative analyses of the -omics data putatively identified genes regulated by DNA methylation. Collectively these data improve our interpretation of how tomato fruit ripening patterns are altered by postharvest practices, and long-term are expected to help improve fruit quality.

plant biology↗

bioRxiv2023

SARS-CoV-2 neurotropism-induced anxiety and depression-like behaviors require Microglia activation

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with a wide range of "long COVID" neurological symptoms. However, the mechanisms governing SARS-CoV-2 neurotropism and its effects on long-term behavioral changes remain poorly understood. Using a highly virulent mouse-adapted SARS-CoV-2 strain, denoted as SARS2-N501YMA30, we demonstrated that intranasal inoculation of SARS2-N501YMA30 results in viral dissemination to multiple brain regions, including the amygdala and hippocampus. Behavioral assays indicated a marked elevation in anxiety- and depression-like behaviors post infection. A comparative analysis of RNA expression profiles disclosed alterations in the post-infected brains. Additionally, we observed dendritic spine remodeling on neurons within the amygdala after infection. Infection with SARS2-N501YMA30 was associated with microglial activation and a subsequent increase in microglia-dependent neuronal activity in the amygdala. Pharmacological inhibition of microglial activity subsequent to viral spike inoculation mitigates microglia-dependent neuronal hyperactivity. Transcriptomic analysis of infected brains revealed the upregulation of inflammatory and cytokine-related pathways, implicating microglia-driven neuroinflammation in the pathogenesis of neuronal hyperactivity and behavioral abnormality. Overall, these data provide critical insights into the neurological consequences of SARS-CoV-2 infection and underscore microglia as a potential therapeutic target for ameliorating virus-induced neurobehavioral abnormalities.

neuroscience↗

bioRxiv2023

HybriSeq: Probe-based Device-free Single-cell RNA Profiling

We have developed the HybriSeq method for single-cell RNA profiling, which utilizes in situ hybridization of multiple probes for targeted transcripts, followed by split-pool barcoding and sequencing analysis of the probes. We have shown that HybriSeq can achieve high sensitivity for RNA detection with multiple probes and profile entire transcripts without an end bias. The utility of HybriSeq is demonstrated in characterizing cell-to-cell heterogeneities of a panel of 196 genes in peripheral blood mononuclear cells and the detection of missed annotations of transcripts.

bioengineering↗

bioRxiv2023

TransGeneSelector: A Transformer-based Approach Tailored for Key Gene Mining with Small Plant Transcriptomic Datasets

Gene mining, particularly from small sample sizes such as in plants, remains a challenge in life sciences. Traditional methods often omit significant genes, while deep learning techniques are hindered by small sample constraints and lack specialized gene mining approaches. This paper presents TransGeneSelector, the first deep learning method tailored for key gene mining in small transcriptomic datasets, ingeniously integrating data augmentation, sample filtering, and a Transformer-based classifier. Tested on Arabidopsis thaliana seeds germination classification using just 79 samples, it not only achieves classification performance on par with, if not superior to, Random Forest and SVM but also excels in identifying upstream regulatory genes that Random Forest might miss, and these pinpointed genes more accurately reflect the metabolic processes inherent in seed germination. TransGeneSelectors ability to mine vital genes from limited datasets signifies its potential as the current state-of-the-art in gene mining in small sample scenarios, providing an efficient and versatile solution for this critical research area.

bioinformatics↗

bioRxiv2023

Nifuroxazide enhances radiotherapy efficacy against hepatocellular carcinoma by upregulating PD-L1 degradation via the ubiquitin-proteasome pathway

Radiation therapy is a primary treatment for hepatocellular carcinoma (HCC), but its effectiveness can be diminished by various factors. The over-expression of PD-L1 has been identified as a critical reason for radiotherapy resistance. Previous studies have demonstrated that nifuroxazide exerts antitumor activity by damaging the Stat3 pathway, but its efficacy against PD-L1 has remained unclear. In this study, we investigated whether nifuroxazide could enhance the efficacy of radiotherapy in HCC by reducing PD-L1 expression. Our results showed that nifuroxazide significantly increased the sensitivity of tumor cells to radiation therapy by inhibiting cell proliferation and migration while increasing apoptosis in vitro. Additionally, nifuroxazide attenuated the up-regulation of PD-L1 expression induced by irradiation, which may be associated with increased degradation of PD-L1 through the ubiquitination-proteasome pathway. Furthermore, nifuroxazide greatly enhanced the efficacy of radiation therapy in H22-bearing mice by inhibiting tumor growth, improving survival, boosting the activation of T lymphocytes, and decelerating the ratios of Treg cells in spleens. Importantly, nifuroxazide limited the increased expression of PD-L1 in tumor tissues induced by radiation therapy. This study confirms, for the first time, that nifuroxazide can augment PD-L1 degradation to improve the efficacy of radiation therapy in HCC-bearing mice.

immunology↗

bioRxiv2023

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states

Schlemms canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemms canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue ([~]4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics. Significance statementSchlemms canal is a critical ocular drainage structure that maintains intraocular pressure with important consequences for glaucoma. Despite this, Schlemms canal has been relatively understudied with the molecular process that mediate its function, maintain its health, or that are compromised in glaucoma needing much deeper characterization. This study is important as it provides the most comprehensive molecular characterization of Schlemms canal cells to date providing a wealth of molecular details. In addition to highlighting important biological pathways, this information will guide studies to develop new treatments against glaucoma.

genomics↗

bioRxiv2023

N4-Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness

A diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N4-hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.

microbiology↗

bioRxiv2023

Analysis of the Neuron-specific IIS/FOXO Transcriptome in Aged Animals Reveals Regulators of Neuronal and Cognitive Aging

Cognitive decline is a significant health concern in our aging society. Here, we used the model organism C. elegans to investigate the impact of the IIS/FOXO pathway on age-related cognitive decline. The daf-2 Insulin/IGF-1 receptor mutant exhibits a significant extension of learning and memory span with age compared to wild-type worms, an effect that is dependent on the DAF-16 transcription factor. To identify possible mechanisms by which aging daf-2 mutants maintain learning and memory with age while wild-type worms lose neuronal function, we carried out neuron-specific transcriptomic analysis in aged animals. We observed downregulation of neuronal genes and upregulation of transcriptional regulation genes in aging wild-type neurons. By contrast, IIS/FOXO pathway mutants exhibit distinct neuronal transcriptomic alterations in response to cognitive aging, including upregulation of stress response genes and downregulation of specific insulin signaling genes. We tested the roles of significantly transcriptionally-changed genes in regulating cognitive functions, identifying novel regulators of learning and memory. In addition to other mechanistic insights, comparison of the aged vs young daf-2 neuronal transcriptome revealed that a new set of potentially neuroprotective genes is upregulated; instead of simply mimicking a young state, daf-2 may enhance neuronal resilience to accumulation of harm and take a more active approach to combat aging. These findings suggest a potential mechanism for regulating cognitive function with age and offer insights into novel therapeutic targets for age-related cognitive decline.

neuroscience↗

bioRxiv2023

ME3BP-7 is a targeted cytotoxic agent that rapidly kills pancreatic cancer cells expressing high levels of monocarboxylate transporter MCT1

Nearly 30% of Pancreatic ductal adenocarcinoma (PDAC)s exhibit a marked overexpression of Monocarboxylate Transporter 1 (MCT1) offering a unique opportunity for therapy. However, biochemical inhibitors of MCT1 have proven unsuccessful in clinical trials. In this study we present an alternative approach using 3-Bromopyruvate (3BP) to target MCT1 overexpressing PDACs. 3BP is a cytotoxic agent that is known to be transported into cells via MCT1, but its clinical usefulness has been hampered by difficulties in delivering the drug systemically. We describe here a novel microencapsulated formulation of 3BP (ME3BP-7), that is effective against a variety of PDAC cells in vitro and remains stable in serum. Furthermore, systemically administered ME3BP-7 significantly reduces pancreatic cancer growth and metastatic spread in multiple orthotopic models of pancreatic cancer with manageable toxicity. ME3BP-7 is, therefore, a prototype of a promising new drug, in which the targeting moiety and the cytotoxic moiety are both contained within the same single small molecule. One Sentence SummaryME3BP-7 is a novel formulation of 3BP that resists serum degradation and rapidly kills pancreatic cancer cells expressing high levels of MCT1 with tolerable toxicity in mice.

cancer biology↗

bioRxiv2023

Engineering cGAS-agonistic oligonucleotides as therapeutics and vaccine adjuvants for cancer immunotherapy

Current cancer immunotherapy (e.g., immune checkpoint blockade (ICB)) has only benefited a small subset of patients. Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) activation holds the potential to improve cancer immunotherapy by eliciting type-I interferon (IFN-I) responses in cancer cells and myeloid cells. Yet, current approaches to this end, mostly by targeting STING, have marginal clinical therapeutic efficacy. Here, we report a cGAS-specific agonistic oligonucleotide, Svg3, as a novel approach to cGAS-STING activation for versatile cancer immunotherapy. Featured with a hairpin structure with consecutive guanosines flanking the stem, Svg3 binds to cGAS and enhances cGAS-Svg3 phase separation to form liquid-like droplets. This results in cGAS activation by Svg3 for robust and dose-dependent IFN-I responses, which outperforms several state-of-the-art STING agonists in murine and human immune cells, and human tumor tissues. Nanocarriers efficiently delivers Svg3 to tissues, cells, and cytosol where cGAS is located. Svg3 reduces tumor immunosuppression and potentiates ICB therapeutic efficacy of multiple syngeneic tumors, in wildtype but neither cGas-/- nor goldenticket Sting-/- mice. Further, as an immunostimulant adjuvant, Svg3 enhances the immunogenicity of peptide antigens to elicit potent T cell responses for robust ICB combination immunotherapy of tumors. Overall, cGAS-agonistic Svg3 is promising for versatile cancer combination immunotherapy.

bioengineering↗

bioRxiv2023

Antisense transcription and PRC2 repression function in parallel during vernalization

Non-coding transcription induces chromatin changes that can mediate environmental responsiveness, but the causes and consequences of these mechanisms are still unclear. Here, we investigate how antisense transcription interfaces with Polycomb Repressive Complex 2 silencing during winter-induced epigenetic regulation of Arabidopsis FLOWERING LOCUS C (FLC). Through genetic, chromatin, and computational analyses, we show that FLC is silenced through pathways that function with different dynamics: an antisense transcription-mediated pathway capable of fast response; and in parallel a slow Polycomb Repressive Complex 2 (PRC2) switching mechanism that maintains each allele in an epigenetically silenced state. Components of both the antisense and PRC2 pathways are regulated by a common transcriptional regulator (NTL8), which accumulates slowly due to reduced growth at low temperatures. The parallel activities of the regulatory steps, which we encapsulate in a mathematical model, creates a flexible system for registering widely fluctuating natural temperature conditions that change year on year, and yet ensure robust epigenetic silencing of FLC. SignificanceThe role of non-coding transcription in establishing and maintaining chromatin states is controversial, mainly because of extensive feedbacks complicating analysis of the relationship between co-transcriptional processing, chromatin state and transcription. This controversy has extended to the role of antisense transcription in the Polycomb-mediated epigenetic silencing of Arabidopsis FLC, a key step in the process of vernalization. Here, we show that antisense transcription and PRC2 silence FLC in parallel pathways that are affected by growth dynamics and temperature fluctuations. These features explain the varied importance of antisense transcription in cold-induced FLC epigenetic silencing seen in various studies using different environmental and growth conditions. The parallel repressive inputs and extensive feedbacks make the mechanism counter-intuitive but provide great flexibility to the plant.

molecular biology↗

bioRxiv2023

Neuronal Activation of the Gαq Protein EGL-30/GNAQ Late in Life Rejuvenates Cognition Across Species

Cognitive decline is perhaps the most devastating aging loss. EGL-30/GNAQ and Gq signaling pathways are highly conserved between C. elegans and mammals. We find that activation of EGL-30 in aged worms at least triples memory span, and we wondered if this highly conserved pathway could also improve memory in very old mice. Murine Gnaq is enriched in hippocampal excitatory neurons and declines with age. Furthermore, GNAQ gain-of-function significantly improved memory in aged mice: GNAQ(gf) in hippocampal neurons of 24-month-old mice rescued age-related impairments in health metrics and long-term memory. Single-nucleus RNAseq revealed gene expression changes related to synaptic function, axon guidance, and learning and memory pathways. Several worm orthologs of mouse genes upregulated by GNAQ(gf) overexpression are required for EGL-30(gf)-dependent memory improvement. These results demonstrate that the molecular and genetic pathways between C. elegans and mammals are highly conserved, as activation of EGL-30/GNAQ, a pathway first identified in worms, rejuvenates cognitive function in two-year old mice (the equivalent of 70-80 yo humans). To our knowledge, this is the oldest age an intervention has successfully improved age-related cognitive decline. One-Sentence SummaryNeuronal activation of the Gq protein EGL-30/GNAQ restores long-term memory at old age in worms and mice.

neuroscience↗

bioRxiv2023

TGFβ controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription

Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease increasing in incidence which disrupts lung health throughout the lifespan. The TGF{beta} superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that Tgfbr2 is critical for AT1 cell fate maintenance and function. Loss of Tgfbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analysis reveal the necessity of Tgfbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF{beta} signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGF{beta} signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

cell biology↗

bioRxiv2023

S2' cleavage site plays a decisive role in expansion of Vero cell tropism by infectious bronchitis virus HV80, with Q855H promoting cell-to-cell fusion

Infectious bronchitis virus (IBV) has restricted cell tropism. Apart from the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered the development of IBV vaccines and research on mechanisms of IBV infection. In a previous study, a new Vero-cell-adapted strain HV80 was obtained via serial chicken embryo and cell passaging of strain H120 and 17 mutations leading to amino acid substitutions occurred in replication gene 1a, S gene and E gene. This study, we constructed recombinants that expressed chimeric S glycoprotein, S1 or S2 subunit of strain H120, and demonstrated that mutations in S2 subunit were related to the Vero cell adaption of strain HV80. With a genome backbone of strain HV80 or H120, and expression of chimeric S2' cleavage site of H120 or HV80, two recombinants demonstrated that the RRRR690/S motif at the S2' cleavage site played a key role in Vero cell adaption of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that F692V enhanced the capability of invasion of HV80 strain, and Q855H induced the formation of syncytia. A transient transfection assay demonstrated different mechanisms for virus-to-cell fusion and cell-to-cell fusion induced by S glycoprotein. The PRRR690/S motif at the S2' cleavage site could be activated by proteases in the process of cell-to-cell fusion, while H855Q substitution did not affect the cell invasion of HV80, but hindered the cell-to-cell fusion by blocking activation of the S2' cleavage site. IMPORTANCEInfectious bronchitis is an acute respiratory disease that has caused large economic losses to the poultry industry. As a member of the gamma-coronaviruses, the restricted cell tropism of infectious bronchitis virus (IBV) limits the development of cellular vaccines and research on infection mechanisms. As a strain that can replicate effectively in mammalian cell lines, studies of HV80s adaptive mechanisms point a way for engineering other IBVs for adaptation in mammalian cell lines. In our study, different recombinants were constructed by reverse genetic techniques, and demonstrated the different mechanism between virus-to-cell and cell-to-cell fusion induced by HV80 S glycoprotein. The acquisition of a highly efficient S2' cleavage site enabled the virus to invade Vero cells. The Q855H substitution played a key role in cell-to-cell fusion, and provided a more efficient model of infection in Vero cells. Our study provides new theoretical insights into mechanisms of IBV adaptation in mammalian cell lines.

microbiology↗

bioRxiv2023

An essential role for miR-15/16 in Treg suppression and restriction of proliferation

The miR-15/16 family is a highly expressed group of tumor suppressor miRNAs that target a large network of genes in T cells to restrict their cell cycle, memory formation and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained immune response. Here, using conditional deletion of miR-15/16 in immunosuppressive regulatory T cells (Tregs) that express FOXP3, we identify new functions of the miR-15/16 family in T cell immunity. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16-deficiency alters Treg expression of critical functional proteins including FOXP3, IL2R/CD25, CTLA4, PD-1 and IL7R/CD127, and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 inhibition of cell cycle programs shifts Treg diversity and produces an effector Treg phenotype characterized by low expression of TCF1, CD25 and CD62L, and high expression of CD44. These Tregs fail to control immune activation of CD4+ effector T cells, leading to spontaneous multi-organ inflammation and increased allergic airway inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance. HighlightsO_LITreg-specific miR-15/16 expression is essential to prevent systemic tissue inflammation C_LIO_LImiR-15/16 restrict Treg proliferation and regulate expression of the key functional Treg molecules FOXP3, IL2R, CTLA4, PD-1 and IL7R C_LIO_LImiR-15/16 limit formation of effector Tregs and is necessary for high suppressive capacity C_LI

molecular biology↗

bioRxiv2023

A systematic search for RNA structural switches across the human transcriptome

RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3UTR of the RORC transcript. In vivo DMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

systems biology↗

bioRxiv2023

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked--that is, phased--is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as linked-reads). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

genomics↗