To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively.However,there is a lack of a p...To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively.However,there is a lack of a pipelined,large-scale,and economical workflow for detecting genome editing outcomes,in particular insertion or deletion of a large fragment.Here,we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products.Recognizing the high error rates of Oxford nanopore sequencing,we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing(GREPore-seq).GREPore-seq can assess nonhomologous end-joining(NHEJ)-mediated double-stranded oligodeoxynucleotide(dsODN)insertions with comparable accuracy to Illumina next-generation sequencing(NGS).GREPore-seq also reveals a full spectrum of homology-directed repair(HDR)-mediated large gene knock-in,correlating well with the fluorescence-activated cell sorting(FACS)analysis results.Of note,we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site.Therefore,we have established a practical workflow to evaluate various genetic changes,including quantifying insertions of short dsODNs,knock-ins of long pieces,plasmid insertions,and large fragment deletions after CRISPR/Cas9-mediated editing.GREPore-seq is freely available at GitHub(https://github.com/lisiang/GREPore-seq)and the National Genomics Data Center(NGDC)BioCode(https://ngdc.cncb.ac.cn/biocode/tools/BT007293).展开更多
Accurate navigation is important for long-range rocket projectile＇s precise striking. To obtain stable and high-per- formance navigation result, a ultra-tight global positioning system/inertial navigation system （GP...Accurate navigation is important for long-range rocket projectile＇s precise striking. To obtain stable and high-per- formance navigation result, a ultra-tight global positioning system/inertial navigation system （GPS/INS） integration based nav- igation approach is proposed. The accurate short-time output of INS is used by GPS receiver to assist in acquisition of signal, and output information of INS and GPS is fused based on federated filter. Meanwhile, the improved cubature Kalman filter with strong tracking ability is chosen to serve as the local filter, and then the federated filter is enhanced based on vector sharing theory. Finally, simulation results show that the navigation accuracy with the proposed method is higher than that with traditional methods. It provides reference for long-range rocket projectile navigation.展开更多
The effects of random long-range connections （shortcuts） on the transitions of neural firing patterns in coupled Hindmarsh-Rose neurons are investigated, where each neuron is subjected to an external current. It is ...The effects of random long-range connections （shortcuts） on the transitions of neural firing patterns in coupled Hindmarsh-Rose neurons are investigated, where each neuron is subjected to an external current. It is found that, on one hand, the system can achieve the transition of neural firing patterns from the fewer-period state to the multi-period one, when the number of the added shortcuts in the neural network is greater than a threshold value, indicating the occurrence of in-transition of neural firing patterns. On the other hand, for a stronger coupling strength, we can also find the similar but reverse results by adding some proper random connections. In addition, the influences of system size and coupling strength on such transition behavior, as well as the internality between the transition degree of firing patterns and its critical characteristics for different external stimulation current, are also discussed.展开更多
本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR produc...本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products)开发出烟草1.8K育种液相芯片(YT1.8K.1)。利用该芯片对上述7份种质两两之间杂交的21个杂交组合进行基因分型检测,每个杂交组合之间的平均差异位点数为650个,能同时满足每个组合定向改良筛选高遗传背景回复率单株的需要。利用该芯片对23个烟草品种进行基因型分型检测和聚类分析,聚类分类结果与品种系谱基本吻合;利用该芯片从367个BC2F1群体中筛选出5个背景回复率高于94.96%的单株,高于理论均值87.5%,表明该育种芯片可应用于烟草种质资源聚类分析、定向改良育种的遗传背景筛选。展开更多
为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38....为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15%~0.23%,批间变异系数为0.09%~0.28%。结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强。临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4%,灵敏度是普通PCR的1 000倍。SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测。展开更多
基金supported by the National Key R&D Program of China(Grant Nos.2016YFA0100600,2019YFA0110800,and 2019YFA0110204)the National Natural Science Foundation of China(Grant Nos.81890990,81730006,81770198,81870149,and 82070115)the Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(CIFMS)(Grant Nos.2019-I2M-1-006 and 2021-I2M-1-041).
文摘To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively.However,there is a lack of a pipelined,large-scale,and economical workflow for detecting genome editing outcomes,in particular insertion or deletion of a large fragment.Here,we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products.Recognizing the high error rates of Oxford nanopore sequencing,we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing(GREPore-seq).GREPore-seq can assess nonhomologous end-joining(NHEJ)-mediated double-stranded oligodeoxynucleotide(dsODN)insertions with comparable accuracy to Illumina next-generation sequencing(NGS).GREPore-seq also reveals a full spectrum of homology-directed repair(HDR)-mediated large gene knock-in,correlating well with the fluorescence-activated cell sorting(FACS)analysis results.Of note,we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site.Therefore,we have established a practical workflow to evaluate various genetic changes,including quantifying insertions of short dsODNs,knock-ins of long pieces,plasmid insertions,and large fragment deletions after CRISPR/Cas9-mediated editing.GREPore-seq is freely available at GitHub(https://github.com/lisiang/GREPore-seq)and the National Genomics Data Center(NGDC)BioCode(https://ngdc.cncb.ac.cn/biocode/tools/BT007293).
基金Project Funded by Chongqing Changjiang Electrical Appliances Industries Group Co.,Ltd
文摘Accurate navigation is important for long-range rocket projectile＇s precise striking. To obtain stable and high-per- formance navigation result, a ultra-tight global positioning system/inertial navigation system （GPS/INS） integration based nav- igation approach is proposed. The accurate short-time output of INS is used by GPS receiver to assist in acquisition of signal, and output information of INS and GPS is fused based on federated filter. Meanwhile, the improved cubature Kalman filter with strong tracking ability is chosen to serve as the local filter, and then the federated filter is enhanced based on vector sharing theory. Finally, simulation results show that the navigation accuracy with the proposed method is higher than that with traditional methods. It provides reference for long-range rocket projectile navigation.
文摘The effects of random long-range connections （shortcuts） on the transitions of neural firing patterns in coupled Hindmarsh-Rose neurons are investigated, where each neuron is subjected to an external current. It is found that, on one hand, the system can achieve the transition of neural firing patterns from the fewer-period state to the multi-period one, when the number of the added shortcuts in the neural network is greater than a threshold value, indicating the occurrence of in-transition of neural firing patterns. On the other hand, for a stronger coupling strength, we can also find the similar but reverse results by adding some proper random connections. In addition, the influences of system size and coupling strength on such transition behavior, as well as the internality between the transition degree of firing patterns and its critical characteristics for different external stimulation current, are also discussed.
文摘本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products)开发出烟草1.8K育种液相芯片(YT1.8K.1)。利用该芯片对上述7份种质两两之间杂交的21个杂交组合进行基因分型检测,每个杂交组合之间的平均差异位点数为650个,能同时满足每个组合定向改良筛选高遗传背景回复率单株的需要。利用该芯片对23个烟草品种进行基因型分型检测和聚类分析,聚类分类结果与品种系谱基本吻合;利用该芯片从367个BC2F1群体中筛选出5个背景回复率高于94.96%的单株,高于理论均值87.5%,表明该育种芯片可应用于烟草种质资源聚类分析、定向改良育种的遗传背景筛选。
文摘为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15%~0.23%,批间变异系数为0.09%~0.28%。结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强。临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4%,灵敏度是普通PCR的1 000倍。SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测。
