Biology subjects

Qian, Y.

Publications and source records attributed to Qian, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Genomic analyses of 10,376 individuals provides comprehensive map of genetic variations, structure and reference haplotypes for Chinese population

Here, we initiated the Westlake BioBank for Chinese (WBBC) pilot project with 4,535 whole-genome sequencing individuals and 5,481 high-density genotyping individuals. We identified 80.99 million SNPs and INDELs, of which 38.6% are novel. The genetic evidence of Chinese population structure supported the corresponding geographical boundaries of the Qinling-Huaihe Line and Nanling Mountains. The genetic architecture within North Han was more homogeneous than South Han, and the history of effective population size of Lingnan began to deviate from the other three regions from 6 thousand years ago. In addition, we identified a novel locus (SNX29) under selection pressure and confirmed several loci associated with alcohol metabolism and histocompatibility systems. We observed significant selection of genes on epidermal cell differentiation and skin development only in southern Chinese. Finally, we provided an online imputation server (https://wbbc.westlake.edu.cn/) which could result in higher imputation accuracy compared to the existing panels, especially for lower frequency variants.

genetics↗

bioRxiv2021

Mechanisms Driving Genome Reduction of a Novel Roseobacter Lineage Showing Vitamin B12 Auxotrophy

Members of the marine Roseobacter group are key players in the global carbon and sulfur cycles. While over 300 species have been described, only 2% possess reduced genomes (mostly 3-3.5 Mbp) compared to an average roseobacter (>4 Mbp). These taxonomic minorities are phylogenetically diverse but form a Pelagic Roseobacter Cluster (PRC) at the genome content level. Here, we cultivated eight isolates constituting a novel Roseobacter lineage which we named CHUG. Metagenomic and metatranscriptomic read recruitment analyses showed that CHUG members were globally distributed and active in marine environments. CHUG members possess some of the smallest genomes (~2.52 Mb) among all known roseobacters, but they do not exhibit canonical features of genome streamlining like higher coding density or fewer paralogues and pseudogenes compared to their sister lineages. While CHUG members are clustered with traditional PRC members at the genome content level, they show important differences. Unlike other PRC members, neither the relative abundances of CHUG members nor their gene expression levels are correlated with chlorophyll a concentration across the global samples. Moreover, CHUG members cannot synthesize vitamin B12, a key metabolite made by most roseobacters but not by many phytoplankton species and thus thought to mediate the roseobacter-phytoplankton interactions. This combination of features is evidence for the hypothesis that CHUG members may have evolved a free-living lifestyle decoupled from phytoplankton. This ecological transition was accompanied by the loss of signature genes involved in roseobacter-phytoplankton symbiosis, suggesting that relaxation of purifying selection is likely an important driver of genome reduction in CHUG.

microbiology↗

bioRxiv2020

Long-range gene regulation network of the MGMT enhancer in modulating glioma cell sensitivity to temozolomide

Acquired resistance to temozolomide (TMZ) is a major obstacle in glioblastoma treatment. MGMT, a DNA repair protein, and the methylation at its gene promoter, plays an important role in TMZ resistance. However, some evidences have suggested a MGMT-independent mechanisms underlying TMZ resistance. Here, we used MGMT enhancer as a model and discovered that its deletion in glioma cells of low MGMT expression induced increased sensitivity to temozolomide. Analysis of a combination of RNA-seq and Capture Hi-C further suggested multiple long-range target genes regulated by the MGMT enhancer and that interactions may play important roles in glioma cell sensitivity to TMZ. This study reveals a novel mechanism of regulation of TMZ sensitivity in glioma cells.

genetics↗

bioRxiv2020

An embryonic zebrafish model to screen disruption of gut-vascular-barriers upon exposure to ambient ultrafine particles

Epidemiological studies have linked exposure to ambient particulate matter (PM) with gastrointestinal (GI) diseases. Ambient ultrafine particle (UFP) are the redox-active sub-fraction of PM2.5, harboring elemental and polycyclic aromatic hydrocarbons from urban environmental sources including diesel and gasoline exhausts. The gut vascular barrier (GVB) regulates paracellular trafficking and systemic disseminations of ingested microbes and toxins. Here, we posit that acute UFP ingestion disrupts the integrity of the intestinal barrier by modulating intestinal Notch activation. Using zebrafish embryos, we performed micro-gavage with the FITC-conjugated dextran (FD10, 10 kDa) to assess the disruption of GVB integrity upon UFP exposure. Following micro-gavage, FD10 retained in the embryonic GI system, migrated through the cloaca. Conversely, co-gavaging UFP increased transmigration of FD10 across the intestinal barrier, and FD10 fluorescence occurred in the venous capillary plexus. Ingestion of UFP further impaired the mid-intestine morphology. We performed micro-angiogram of FD10 to corroborate acute UFP-mediated disruption of GVB. Transient genetic and pharmacologic manipulations of global Notch activity suggested Notch regulation of the GVB. Overall, our integration of a genetically tractable embryonic zebrafish and micro-gavage technique provided epigenetic insights underlying ambient UFP ingestion disrupts the GVB. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/359976v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1430b21org.highwire.dtl.DTLVardef@8fcc2eorg.highwire.dtl.DTLVardef@a5fe0borg.highwire.dtl.DTLVardef@1c9a034_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Presence and Transmission of Mitochondrial Heteroplasmic Mutations in Human Populations of European and African Ancestry

We investigated the concordance of mitochondrial DNA heteroplasmic mutations (heteroplasmies) in different types of maternal pairs (n=6,745 pairs) of European (EA, n=4,718 pairs) and African (AA, n=2,027 pairs) Americans with whole genome sequences (WGSs). The average concordance rate of heteroplasmies was highest between mother-offspring pairs, followed by sibling-sibling pairs and more distantly related maternal pairs in both EA and AA participants. The allele fractions of concordant heteroplasmies exhibited high correlation (R2=0.8) between paired individuals. Compared to concordant heteroplasmies, discordant ones were more likely to locate in coding regions, be nonsynonymous or nonsynonymous-deleterious (p<0.001). The average number of heteroplasmies per individual (i.e. heteroplasmic burden) was at a similar level until older age (70-80 years old) and increased significantly thereafter (p<0.01). The burden of deleterious heteroplasmies (combined annotation-dependent depletion score[≥]15), however, was significantly correlated with advancing age (20-44, 45-64, [≥]65 years, p-trend=0.01). A genome-wide association analysis of the heteroplasmic burden identified many significant (P<5e-8) common variants (minor allele frequency>0.05) at 11p11.12. Many of the top SNPs act as strong long-range cis regulators of protein tyrosine phosphatase receptor type J. This study provides further evidence that mtDNA heteroplasmies may be inherited or somatic. Somatic heteroplasmic variants increase with advancing age and are more likely to have an adverse impact on mitochondrial function. Further studies are warranted for functional characterization of the deleterious heteroplasmies occurring with advancing age and the association of the 11p11.12 region of the nuclear genome with mtDNA heteroplasmy.

genetics↗

bioRxiv2020

MicroRNA variants and HLA-miRNA interactions are novel rheumatoid arthritis susceptibility factors

ObjectiveAlthogh Genome-wide association studies have identified >100 variants for rheumatoid arthritis (RA),the reported genetic variants only explain <40% of RA heritability. We conducted a systemic association study between common East-Asian miRNA SNPs with RA in a large Han Chinese cohort to explain missing heritability and identify miRNA epistatic interactions. Methods4 HLA SNPs (HLA-DRB1, HLA-DRB9, HLA-DQB1 and TNFAIP3) and 225 common SNPs located in miRNA which might influence the miRNA target binding or pre-miRNA stability were genotyped in 1,607 rheumatoid arthritis and 1,580 matched normal individuals. A meta-analysis with previous GWAS studies (4,873 RA cases and 17,642 controls) was performed to discovery another novel miRNA RA-associated SNPs. Results2 novel SNPs including rs1414273 (miR-548ac, OR=0.84, P=8.26x10-4) and rs2620381 (miR-627, OR=0.77, P=2.55x10-3) conferred significant association with RA. Individuals carried 8 risk alleles showed 15.38 (95%CI: 4.69-50.49, P<1.0x10-6) times more risk to be affected by RA. In addition, rs5997893 (miR-3928) showed significant epistasis effect with rs4947332 (HLA-DRB1, OR=4.23, P=0.04) and rs2967897 (miR-5695) with rs7752903 (TNFAIP3, OR=4.43, P=0.03). Finally, we demonstrated targets of the significant miRNAs showed enrichment in immune related genes (P=2.0x10-5) and FDA approved drug target genes (P=0.014). Conclusions6 novel miRNA SNPs including rs1414273 (miR-548ac, P=8.26x10-4), rs2620381 (miR-627, P=2.55x10-3), rs4285314 (miR-3135b, P=1.10x10-13), rs28477407 (miR-4308, P=3.44x10-5), rs5997893 (miR-3928, P=5.9x10-3) and rs45596840 (miR-4482, P=6.6x10-3) were confirmed to be significantly associated with RA in a Chinese population. Our study suggests that miRNAs might be interesting targets to accelerate the understanding of the pathogenesis and drug development for rheumatoid arthritis.

genetics↗

bioRxiv2020

Simultaneous transcriptome and methylome profiles of single mouse oocytes provide novel insights on maturation and aging

BackgroundAdvanced maternal aging has become a worldwide public health issue that contributes to female fertility decline and significant risk to embryo development. Despite transcriptional and epigenetic alterations reported in oocyte maturation and development, the dynamics of gene expression and DNA dynamics associated with aging remain elusive. Here we generated simultaneous transcriptome and methylome profiles of mouse oocytes during aging and maturation at single-cell and single-base resolution to examine key biological processes and identify the key targets for novel treatment options. ResultsWe report the dynamics in transcriptome and DNA methylome in mouse oocytes during maternal aging and oocyte maturation. Age-associated gene expression changes showed mitochondrial dysfunction in GV oocytes and defects of chromosome segregation and spindle assembly in MII oocytes. EIF2 signaling protein synthesis pathway was also impaired during aged oocyte maturation. Moreover, distinctive DNA methylation patterns were demonstrated during maternal aging in GV and MII oocytes. A positive correlation between gene expression and methylation in gene body was characterized. Furthermore, we identified several promising biomarkers, including IL-7, to assess oocyte quality, which are potential therapeutic targets for improve oocyte maturation. More importantly, we built the first mouse oocyte maturation and age prediction model using transcriptome data and validated its feasibility in published data. ConclusionsThis work provides a better understanding of molecular and cellular mechanisms during mouse oocyte aging, points a new direction of oocyte quality assessment, and paves the way for developing novel treatments to improve oocyte maturation and quality in the future.

genomics↗

bioRxiv2020

Temporal Metabolic Characteristics and Transcriptomic Landscape of Islets and Liver Reveal Dynamic Pathophysiology and Interorgan Crosstalk in High-fat Diet-induced Diabetes

ObjectiveHyperinsulinemia and insulin resistance are co-existing characteristics of type 2 diabetes, whereas the molecular mechanism underlying this deleterious cycle remains elusive. The temporal transcriptomic landscape of core organs responsible for insulin secretion (islets) and insulin action (liver) could provide new insights. MethodsThe longitudinal profiling of glucose metabolism, insulin sensitivity, islet architecture and secretion were conducted in C57BL/6N mice fed a high-fat diet (HFD) or chow diet for 24 weeks. RNA-sequencing of islets and liver were performed once every 4 weeks. Weighted gene co-expression network analysis and Ingenuity Pathway Analysis were applied to construct networks and evaluate co-ordinated molecular interactions between islets and liver. ResultsMice exhibited progressively deteriorated glucose homeostasis with hyperinsulinemia but impaired first-phase insulin secretion after 4 weeks on HFD. Insulin, glucagon and somatostatin secretion in response to glucose with or without palmitate gradually deteriorated from dysregulation to failure. Systemic insulin resistance developed over 24 weeks with variable time course in tissue-specific insulin action. Our transcriptomic datasets outlined the impact of HFD on dynamics of islet and liver molecular network at different stages. Correlation analyses revealed that both organs jointly programmed {beta}-cell compensatory adaption via cell proliferation at early phase and irreversible islet dysfunction by inappropriate immune response at later stage. Alternations of T cell subpopulations validated the participation of adaptive immune response through priming and amplification phases in diabetic progression. ConclusionOur data provide a comprehensive landscape of crosstalk between islets and liver in diet-induced diabetes, elucidating the development of islet dysfunction and insulin resistance. HighlightsO_LIDiet-induced diabetes is featured by transition from islet dysfunction to failure C_LIO_LIInsulin resistance develops with variable time course in different tissues C_LIO_LIDynamics of islet and liver molecular network interplay at different stages C_LIO_LICell proliferation and improper immune reaction mediated interorgan crosstalk C_LIO_LIAdaptive immune response participated via priming and amplification phases C_LI

cell biology↗

bioRxiv2020

dCas9 regulator to neutralize competition in CRISPRi circuits

CRISPRi-mediated gene repression allows simultaneous control of many genes. However, despite highly specific sgRNA-promoter binding, multiple sgRNAs still interfere with one another by competing for dCas9. We created a dCas9 regulator that adjusts dCas9 concentration based on sgRNAs demand, mitigating competition in CRISPRi-based logic gates. The regulators performance is demonstrated on both single-stage and layered CRISPRi logic gates and in two common E. coli strains. When a competitor sgRNA causes between two and ~25 fold-change in a logic gates input/output response without dCas9 regulator, the response is essentially unchanged when the regulator is used. The dCas9 regulator thus enables concurrent and independent operation of multiple sgRNAs, thereby supporting independent control of multiple genes.

synthetic biology↗

bioRxiv2020

Molecular evolution of SARS-CoV-2 structural genes: evidence of positive selection in spike glycoprotein

SARS-CoV-2 caused a global pandemic in early 2020 and has resulted in more than 8,000,000 infections as well as 430,000 deaths in the world so far. Four structural proteins, envelope (E), membrane (M), nucleocapsid (N) and spike (S) glycoprotein, play a key role in controlling the entry into human cells and virion assembly of SARS-CoV-2. However, how these genes evolve during its human to human transmission is largely unknown. In this study, we screened and analyzed roughly 3090 SARS-CoV-2 isolates from GenBank database. The distribution of the four gene alleles is determined:16 for E, 40 for M, 131 for N and 173 for S genes. Phylogenetic analysis shows that global SARS-CoV-2 isolates can be clustered into three to four major clades based on the protein sequences of these genes. Intragenic recombination event isnt detected among different alleles. However, purifying selection has conducted on the evolution of these genes. By analyzing full genomic sequences of these alleles using codon-substitution models (M8, M3 and M2a) and likelihood ratio tests (LRTs) of codeML package, it reveals that codon 614 of S glycoprotein has subjected to strong positive selection pressure and a persistent D614G mutation is identified. The definitive positive selection of D614G mutation is further confirmed by internal fixed effects likelihood (IFEL) and Evolutionary Fingerprinting methods implemented in Hyphy package. In addition, another potential positive selection site at codon 5 in the signal sequence of the S protein is also identified. The allele containing D614G mutation has undergone significant expansion during SARS-CoV-2 global pandemic, implying a better adaptability of isolates with the mutation. However, L5F allele expansion is relatively restricted. The D614G mutation is located at the subdomain 2 (SD2) of C-terminal portion (CTP) of the S1 subunit. Protein structural modeling shows that the D614G mutation may cause the disruption of salt bridge among S protein monomers increase their flexibility, and in turn promote receptor binding domain (RBD) opening, virus attachment and entry into host cells. Located at the signal sequence of S protein as it is, L5F mutation may facilitate the protein folding, assembly, and secretion of the virus. This is the first evidence of positive Darwinian selection in the spike gene of SARS-CoV-2, which contributes to a better understanding of the adaptive mechanism of this virus and help to provide insights for developing novel therapeutic approaches as well as effective vaccines by targeting on mutation sites.

evolutionary biology↗

bioRxiv2020

A system-view of B. pertussis booster vaccine responses in adults primed with whole-cell vs. acellular vaccine in infancy

Whole-cell inactivated vaccine against Bordetella pertussis (wP) was substituted in many countries by an acellular subunit vaccine (aP) to reduce side effects. Recent epidemiological studies have shown that aP vaccination in infancy induces less durable immunity than wP vaccination. To determine immunological differences associated with aP vs. wP priming, we performed system-level profiling of the immune response in adults primed with aP vs. wP vaccine in infancy following the Tdap booster vaccination as a surrogate to antigen encounter in vivo. Shared immune responses across cohorts were identified, including an increase of the blood monocyte frequency on day 1, and strong antigen-specific IgG response seven days after boost. Comparing aP and wP primed individuals, we found a subset of aP-primed individuals with higher levels of expression for several genes including CCL3 on day 3 and NFKBIA and ICAM1 on day 7 post immunization. These observations were supported by increased CCL3 concentrations in plasma of aP primed individuals. Contrary to the wP individuals, the CCL3-high aP subset presented boosted PT-specific IgE responses. Furthermore, higher antigen specific IgG4 and IgG3 antibodies against specific vaccine antigens at baseline and post boost of aP individuals was observed, suggesting a long term maintained difference in the IgG subtype response. Overall our findings demonstrate that, while broad immune response patterns to Tdap boost overlap between aP and wP primed individuals, a subset of aP primed individuals present a divergent response. These findings provide candidate targets to study the causes and correlates of waning immunity after aP vaccination.

systems biology↗

bioRxiv2020

Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging

Near-infrared (NIR) genetically-encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross-talk with visible-light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report two improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and C. elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

molecular biology↗

bioRxiv2020

The Nucleocapsid Protein of SARS-CoV-2 Abolished Pluripotency in Human Induced Pluripotent Stem Cells

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging across the world, leading to a global mortality rate of 3.4% (estimated by World Health Organization in March 2020). As a potential vaccine and therapeutic target, the nucleocapsid protein of SARS-CoV-2 (nCoVN) functions in packaging the viral genome and viral self-assembly. To investigate the biological effects of nCoVN to human stem cells, genetically engineered human induced pluripotent stem cells (iPSC) expressing nCoVN (iPSC-nCoVN) were generated by lentiviral expression systems, in which the expression of nCoVN could be induced by the doxycycline. The proliferation rate of iPSC-nCoVN was decreased. Unexpectedly, the morphology of iPSC started to change after nCoVN expression for 7 days. The pluripotency marker TRA-1-81 were not detectable in iPSC-nCoVN after a four-day induction. Meanwhile, iPSC-nCoVN lost the ability for differentiation into cardiomyocytes with a routine differentiation protocol. The RNA-seq data of iPSC-nCoVN (induction for 30 days) and immunofluorescence assays illustrated that iPSC-nCoVN were turning to fibroblast-like cells. Our data suggested that nCoVN disrupted the pluripotent properties of iPSC and turned them into other types of cells, which provided a new insight to the pathogenic mechanism of SARS-CoV-2.

cell biology↗

bioRxiv2020

APOL1 Variant-Expressing Endothelial Cells Exhibit Autophagic Dysfunction and Mitochondrial Stress

Apolipoprotein L1 (APOL1) gene risk variants (RV) associate with renal and cardiovascular disease particularly in SLE. We hypothesized that in RV-carrying human umbilical vein endothelial cells (HUVECs) cytokine-induced APOL1 expression compromises mitochondrial respiration, lysosome integrity, and autophagic flux. HUVEC cultures of each APOL1 genotype were generated. APOL1 was expressed using IFN{gamma}; HUVEC mitochondrial function, lysosome integrity, and autophagic flux were measured. IFN{gamma} increased APOL1 expression across all genotypes 20-fold (p=0.001). Compared to the homozygous G0 (ancestral) allele (0RV), high risk (2RV) HUVECs showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosome compartment (p<0.001), and an accumulation of autophagosomes suggesting a defect in autophagic flux. Treatment of 0RV HUVECs with a non-selective lysosome inhibitor, hydroxychloroquine, produced autophagosome accumulations similar to the 2RV cells, thus implicating lysosome dysfunction in blocking autophagy. Compared to 0RV and 2RV HUVECs, 1 RV cells demonstrated an intermediate autophagy defect which was exacerbated by IFN{gamma}. Our findings implicate dysfunction of mitochondrial respiration, lysosome, and autophagy in APOL1 RV-mediated endothelial cytotoxicity. IFN{gamma} amplified this phenotype even in variant heterozygous cells-a potential underpin of the APOL1/inflammation interaction. This is the first description of APOL1 pathobiology in variant heterozygous cell cultures.

cell biology↗

bioRxiv2020

Tissue engineered model of hepatic breast cancer micrometastasis shows host-dependent colonization patterns and drug responses

Early stages of colonization of distant organs by metastatic cancer cells (micrometastasis) remain almost inaccessible to study due to lack of relevant experimental approaches. Here, we show the first 3D tissue engineered model of hepatic micrometastasis of triple negative breast cancer (TNBC). It reproduces characteristic histopathological features of the disease and reveals that metastatic TNBC cells colonize liver parenchymal and stromal extracellular matrix with different speed and by different strategies. These engineered tumors induce the angiogenic switch when grafted in vivo, confirming their metastatic-specific behaviour. Furthermore, we proved feasibility and biological relevance of our model for drug and nanoparticle testing and found a down-regulatory effect of the liver microenvironment of the sensitivity of TNBC cells to chemotherapeutic drug doxorubicin in free and nanoformulated forms. The convenient and affordable methodology established here can be translated to other types of metastatic tumors for basic cancer biology research and adapted for high-throughput assays.

bioengineering↗

bioRxiv2019

An endoribonuclease-based incoherent feedforward loop for decoupling resource-limited genetic modules

Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a devices output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is competition among genes for shared cellular resources, such as those required for transcription and translation, which can induce coupling among otherwise independently-regulated genes. Here, we quantify the effects of resource sharing on engineered genetic systems in mammalian cells and develop an endoribonuclease-based incoherent feedforward loop (iFFL) to make gene expression levels robust to changes in resource availability. Our iFFL accurately controls gene expression levels in various cell lines and in the presence of significant resource sequestration by transcriptional activators. In addition to mitigating resource sharing, our iFFL also adapts gene expression to multiple log decades of DNA copy number variation, substantially improving upon previously-described miRNA-based iFFLs. Ultimately, our iFFL device will enable predictable, robust, and context-independent control of gene expression in mammalian cells.

synthetic biology↗

bioRxiv2019

Notch signaling is required for survival of the germline stem cell lineage in testes of Drosophila melanogaster

In all metazoan species, sperm is produced from germline stem cells. These self-renew and produce daughter cells that amplify and differentiate dependent on interactions with somatic support cells. In the male gonad of Drosophila melanogaster, the germline and somatic cyst cells co-differentiate as cysts, an arrangement in which the germline is completely enclosed by cytoplasmic extensions from the cyst cells. Notch is a developmentally relevant receptor in a pathway requiring immediate proximity with the signal sending cell. Here, we show that Notch is expressed in the cyst cells of wild-type testes. Notch becomes activated in the transition zone, an apical area of the testes in which the cyst cells express stage-specific transcription factors and the enclosed germline finalizes transit-amplifying divisions. Reducing the ligand Delta from the germline cells via RNA-Interference or reducing the receptor Notch from the cyst cells via CRISPR resulted in cell death concomitant with loss of germline cells from the transition zone. This shows that Notch signaling is essential for the survival of the germline stem cell lineage.

developmental biology↗

bioRxiv2019

Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

The major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

genomics↗