Biology subjects

Yang, H.

Publications and source records attributed to Yang, H..

695 records · Page 39Linked to original sources

bioRxiv2018

Circularization of genes and chromosome by CRISPR in human cells

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders and diseases such as cancer. However, there is a lack of genetic engineering tool to recapitulate these features. Here, we report the discovery that delivery of pairs of CRISPR/Cas9 guide RNAs into human cells generate functional eccDNAs and ring chromosomes. We generated a dual-fluorescence eccDNA biosensor system, which allows us to study CRISPR deletion, inversion, and circularization of genes inside cells. Analysis after CRISPR editing at intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts reveal that CRISPR deleted DNA readily form eccDNA in human cells. DNA in sizes from a few hundred base pairs up to a 47.4 megabase-sized ring chromosome (chr18) can be circularized. Our discoveries advance and expand CRISPR-Cas9 technology applications for genetic engineering, modeling of human diseases, and chromosome engineering.\n\nOne Sentence Summary: CRISPR circularization of DNA offers new tools for studying eccDNA biogenesis, function, chromosome engineering, and synthetic biology.

genomics↗

bioRxiv2018

Open-pFind enables precise, comprehensive and rapid peptide identification in shotgun proteomics

Shotgun proteomics has grown rapidly in recent decades, but a large fraction of tandem mass spectrometry (MS/MS) data in shotgun proteomics are not successfully identified. We have developed a novel database search algorithm, Open-pFind, to efficiently identify peptides even in an ultra-large search space which takes into account unexpected modifications, amino acid mutations, semi- or non-specific digestion and co-eluting peptides. Tested on two metabolically labeled MS/MS datasets, Open-pFind reported 50.5-117.0% more peptide-spectrum matches (PSMs) than the seven other advanced algorithms. More importantly, the Open-pFind results were more credible judged by the verification experiments using stable isotopic labeling. Tested on four additional large-scale datasets, 70-85% of the spectra were confidently identified, and high-quality spectra were nearly completely interpreted by Open-pFind. Further, Open-pFind was over 40 times faster than the other three open search algorithms and 2-3 times faster than three restricted search algorithms. Re-analysis of an entire human proteome dataset consisting of [~]25 million spectra using Open-pFind identified a total of 14,064 proteins encoded by 12,723 genes by requiring at least two uniquely identified peptides. In this search results, Open-pFind also excelled in an independent test for false positives based on the presence or absence of olfactory receptors. Thus, a practical use of the open search strategy has been realized by Open-pFind for the truly global-scale proteomics experiments of today and in the future.

bioinformatics↗

bioRxiv2018

A catalog of microbial genes from the bovine rumen reveals the determinants of herbivory

BackgroundThe rumen microbiota provides essential services to its host and, through its role in ruminant production, contributes to human nutrition and food security. A thorough knowledge of the genetic potential of rumen microbes will provide opportunities for improving the sustainability of ruminant production systems. The availability of gene reference catalogs from gut microbiomes has advanced the understanding of the role of the microbiota in health and disease in humans and other mammals. In this work, we established a catalog of reference prokaryote genes from the bovine rumen.\n\nResultsUsing deep metagenome sequencing we identified 13,825,880 non-redundant prokaryote genes from the bovine rumen. Compared to human, pig and mouse gut metagenome catalogs, the rumen is larger and richer in functions and microbial species associated with the degradation of plant cell wall material and production of methane. Genes encoding enzymes catalyzing the breakdown of plant polysaccharides showed a particularly high richness that is otherwise impossible to infer from available genomes or shallow metagenomics sequencing. The catalog expands by several folds the dataset of carbohydrate-degrading enzymes described in the rumen. Using an independent dataset from a group of 77 cattle fed 4 common dietary regimes, we found that only <0.1% of genes were shared by all animals, which contrast with a large overlap for functions, i.e. 63% for KEGG functions. Different diets induced differences in the relative abundance rather than the presence or absence of genes explaining the great adaptability of cattle to rapidly adjust to dietary changes.\n\nConclusionsThese data bring new insights into functions, carbohydrate-degrading enzymes and microbes of the rumen that is complementing the available information on microbial genomes. The catalog is a significant biological resource enabling deeper understanding of phenotypes and biological processes and will be expanded as new data is made available.

microbiology↗

bioRxiv2018

Identification of Variable and Joining germline genes and alleles for Rhesus macaque from B-cell receptor repertoires

The Rhesus macaque is a valuable preclinical animal model to estimate vaccine effectiveness, and is also important for understanding antibody maturation and B-cell repertoire evolution responding to vaccination; however, incomplete mapping of rhesus immunoglobulin germline genes hinders the research efforts. To address this deficiency, we sequenced B-cell receptor (BCR) repertoires of 75 India Rhesus macaques. Using a bioinformatic method that has been validated with BCR repertoire analysis of three human donors, we were able to infer rhesus Variable (V) and Joint(J) germline alleles, identifying a total of 122 V and 20 J germline alleles. Importantly, 91 V and 13 J alleles were novel, and 40 V and 13 J genes were found at a novel genome region that has not been previously recorded. The novelty of these newly identified alleles was supported by two observations. Firstly, 50 V and 5 J novel alleles were observed in whole genome sequencing data of 10 Rhesus macaques. Secondly, using alignment reference including the novel alleles, the mutation rate of rearranged repertoires was significant declined in 9 other irrelevant samples, and all our identified novel V and J alleles were 100% identity mapped by rearranged repertoire data. These newly identified novel alleles, along with previous reported alleles, provide an important reference for future investigations of rhesus immune repertoire evolution, in response to vaccination or infection. In addition, the method outlined in our study offered an example to future efforts in identifying novel immunoglobulin alleles.

immunology↗

bioRxiv2018

Generation of a novel growth-enhanced and reduced environmental impact transgenic pig strain

In pig production, insufficient feed digestion causes excessive nutrients such as phosphorus and nitrogen, which are then released to the environment. To address the issue of environmental emissions, we have established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, {beta}-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphate outputs were reduced by 23%-46%, and growth rate improved by 23.4% (gilts) and 24.4% (boars) when the pigs were fed on a corn and soybean-based diet and high-NSP diet. The transgenic pigs showed a 11.5%- 14.5% improvement in feed conversion rate compared to the age-matched wild-type littermates. These findings indicate that transgenic pigs are promising resources for improving feed efficiency and reducing nutrient emissions to the environment.

biochemistry↗

bioRxiv2017

Dynamic clustering of dynamin-amphiphysin rings regulates membrane constriction and fission coupled with GTP hydrolysis

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin mediated endocytosis. Dynamin forms washer ring-shaped/helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulates membrane remodeling during fission, but its precise mechanism remains elusive. In this study, we analyze structural changes of dynamin-amphiphysin complexes during membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin rings are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our new approaches using combination of EM and HS-AFM clearly demonstrates dynamics of dynamin-amphiphysin complexes during membrane fission suggesting a novel \"clusterase\" model of dynamin-mediated membrane fission.

biophysics↗

bioRxiv2017

Systematic mapping of chromatin state landscapes during mouse development

Embryogenesis requires epigenetic information that allows each cell to respond appropriately to developmental cues. Histone modifications are core components of a cells epigenome, giving rise to chromatin states that modulate genome function. Here, we systematically profile histone modifications in a diverse panel of mouse tissues at 8 developmental stages from 10.5 days post conception until birth, performing a total of 1,128 ChIP-seq assays across 72 distinct tissue-stages. We combine these histone modification profiles into a unified set of chromatin state annotations, and track their activity across developmental time and space. Through integrative analysis we identify dynamic enhancers, reveal key transcriptional regulators, and characterize the role of chromatin-based repression in developmental gene regulation. We also leverage these data to link enhancers to putative target genes, revealing connections between coding and non-coding sequence variation in disease etiology. Our study provides a compendium of resources for biomedical researchers, and achieves the most comprehensive view of embryonic chromatin states to date.

genomics↗

bioRxiv2017

Whole exome sequencing study of colorectal cancer in Chinese population reveals novel prevalently mutated genes and decreased mutation frequency of APC and Wnt signaling in lymph node positive cancer

Colorectal cancer is the fifth prevalent cancer in China. Nevertheless, a large-scale characterization of Chinese colorectal cancer mutation spectrum has not been carried out. In this study, we have performed whole exome-sequencing analysis of 98 patients tumor samples with matched pairs of normal colon tissues using Illumina and Complete Genomics high-throughput sequencing platforms. Canonical CRC somatic gene mutations with high prevalence (>10%) have been verified, including TP53, APC, KRAS, SMAD4, FBXW7 and PIK3CA. PEG3 is identified as a novel frequently mutated gene (10.6%). APC and Wnt signaling exhibit significantly lower mutation frequencies than those in TCGA data. Analysis with clinical characteristics indicates that APC gene and Wnt signaling display lower mutation rate in lymph node positive cancer than negative ones, which are not observed in TCGA data. APC gene and Wnt signaling are considered as the key molecule and pathway for colorectal cancer initiation, and these findings greatly undermine their importance in tumor progression for Chinese patients. Taken together, the application of next-generation sequencing has led to the determination of novel somatic mutations and alternative disease mechanisms in colorectal cancer progression, which may be useful for understanding disease mechanism and personalizing treatment for Chinese patients.

genomics↗

bioRxiv2017

Olfactory Receptors Are Required For Social Behavior And Neural Plasticity In Ants, As Evidenced By CRISPR-Mediated Gene Knockout

The chemosensory system is key to establishing and maintaining social structure in eusocial insects. Ants exhibit cooperative colonial behaviors reflective of an advanced form of sociality with an extensive dependency on communication. Cuticular hydrocarbons (CHCs) serve as pheromones and cues that regulate multiple aspects of social interactions and behaviors in ants. The perception of CHCs entails odorant receptor neurons (ORNs) that express specific odorant receptors (ORs) encoded by a dramatically expanded Or gene family in ants. Until recently, studies of the biological functions of ORs in eusocial insects were stymied by the lack of genetic tools. In most eusocial insect species, only one or a few queens in a colony can transmit the genetic information to their progeny. In contrast, any worker in the ant Harpegnathos saltator can be converted into a gamergate (pseudo-queen), and used as a foundress to engender an entire new colony and be crossed for genetic experiments. This feature facilitated CRISPR-Cas9 gene targeting to generate a germline mutation in the orco gene that encodes the obligate co-receptor whose mutation should significantly impact ant olfaction. Our results show that Orco exhibits a conserved role in the perception of general odorants but also a role in reproductive physiology and social behavior plasticity in ants. Surprisingly, and in contrast to other insect systems, the loss of OR functionality also dramatically reduces the development of the ant antennal lobe where ORNs project. Taken together, these findings open the possibility of studying the genetics of eusociality and provide inroads towards understanding the function of the expanded ORs family in eusocial insects in regulating caste determination, social communication and neuronal plasticity.

neuroscience↗

bioRxiv2017

HadoopCNV: A Dynamic Programming Imputation Algorithm To Detect Copy Number Variants From Sequencing Data

BACKGROUNDWhole-genome sequencing (WGS) data may be used to identify copy number variations (CNVs). Existing CNV detection methods mostly rely on read depth or alignment characteristics (paired-end distance and split reads) to infer gains/losses, while neglecting allelic intensity ratios and cannot quantify copy numbers. Additionally, most CNV callers are not scalable to handle a large number of WGS samples.\n\nMETHODSTo facilitate large-scale and rapid CNV detection from WGS data, we developed a Dynamic Programming Imputation (DPI) based algorithm called HadoopCNV, which infers copy number changes through both allelic frequency and read depth information. Our implementation is built on the Hadoop framework, enabling multiple compute nodes to work in parallel.\n\nRESULTSCompared to two widely used tools - CNVnator and LUMPY, HadoopCNV has similar or better performance on both simulated data sets and real data on the NA12878 individual. Additionally, analysis on a 10-member pedigree showed that HadoopCNV has a Mendelian precision that is similar or better than other tools. Furthermore, HadoopCNV can accurately infer loss of heterozygosity (LOH), while other tools cannot. HadoopCNV requires only 1.6 hours for a human genome with 30X coverage, on a 32-node cluster, with a linear relationship between speed improvement and the number of nodes. We further developed a method to combine HadoopCNV and LUMPY result, and demonstrated that the combination resulted in better performance than any individual tools.\n\nCONCLUSIONSThe combination of high-resolution, allele-specific read depth from WGS data and Hadoop framework can result in efficient and accurate detection of CNVs.

bioinformatics↗

bioRxiv2017

An Integrative Framework For Detecting Structural Variations In Cancer Genomes

Structural variants can contribute to oncogenesis through a variety of mechanisms, yet, despite their importance, the identification of structural variants in cancer genomes remains challenging. Here, we present an integrative framework for comprehensively identifying structural variation in cancer genomes. For the first time, we apply next-generation optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole genome sequencing to systematically detect SVs in a variety of cancer cells.\n\nUsing this approach, we identify and characterize structural variants in up to 29 commonly used normal and cancer cell lines. We find that each method has unique strengths in identifying different classes of structural variants and at different scales, suggesting that integrative approaches are likely the only way to comprehensively identify structural variants in the genome. Studying the impact of the structural variants in cancer cell lines, we identify widespread structural variation events affecting the functions of non-coding sequences in the genome, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel 3D chromatin structural domains.\n\nThese results underscore the importance of comprehensive structural variant identification and indicate that non-coding structural variation may be an underappreciated mutational process in cancer genomes.

genomics↗