[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 gen...[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.展开更多
Type 2 diabetes(T2D)mellitus is a common complex disease that currently affects more than 400 million people worldwide and has become a global health problem.High-throughput sequencing technologies such as whole-genom...Type 2 diabetes(T2D)mellitus is a common complex disease that currently affects more than 400 million people worldwide and has become a global health problem.High-throughput sequencing technologies such as whole-genome and whole-exome sequencing approaches have provided numerous new insights into the molecular bases of T2D.Recent advances in the application of sequencing technologies to T2D research include,but are not limited to:(1)Fine mapping of causal rare and common genetic variants;(2)Identification of confident genelevel associations;(3)Identification of novel candidate genes by specific scoring approaches;(4)Interrogation of disease-relevant genes and pathways by transcriptional profiling and epigenome mapping techniques;and(5)Investigation of microbial community alterations in patients with T2D.In this work we review these advances in application of next-generation sequencing methods for elucidation of T2D pathogenesis,as well as progress and challenges in implementation of this new knowledge about T2D genetics in diagnosis,prevention,and treatment of the disease.展开更多
AIM：To summarize the phenotypes and identify the underlying genetic cause of the CRYBB1 and CRYBB2 gene responsible for congenital cataract in two Chinese families.METHODS：Detailed family histories and clinical data...AIM：To summarize the phenotypes and identify the underlying genetic cause of the CRYBB1 and CRYBB2 gene responsible for congenital cataract in two Chinese families.METHODS：Detailed family histories and clinical data were collected from patients during an ophthalmologic examination. Of 523 inheritable genetic vision systemrelated genes were captured and sequenced by targeted next-generation sequencing,and the results were confirmed by Sanger sequencing. The possible functional impacts of an amino acid substitution were performed with Poly Phen-2 and SIFT predictions.RESULTS：The patients in the two families were affected with congenital cataract. Sixty-five （FAMILY-1） and sixty two （FAMILY-2） single-nucleotide polymorphisms and indels were selected by recommended filtering criteria.Segregation was then analyzed by applying Sanger sequencing with the family members. A heterozygous CRYBB1 mutation in exon 4 （c.347T〉C, p.L116P） was identified in sixteen patients in FAMILY-1. A heterozygous CRYBB2 mutation in exon 5 （c.355G〉A, p.G119R） was identified in three patients in FAMILY-2. Each mutation cosegregated with the affected individuals and did not exist in unaffected family members and 200 unrelated normal controls.The mutation was predicted to be highly conservative and to be deleterious by both PolyPhen-2 and SIFT.CONCLUSION：TheCRYBB1 mutation（c.347T〉C）and CRYBB2 mutation （c.355G〉A） are novel in patients with congenital cataract. We summarize the variable phenotypes among the patients, which expanded the phenotypic spectrum of congenital cataract in a different ethnic background.展开更多
Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing.While the rapid acquisition of SARS-Co V-2 genome from clini...Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing.While the rapid acquisition of SARS-Co V-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background.To address this issue,we modified and evaluated an approach by utilizing SARS-Co V-2-specific amplicon amplification and Oxford Nanopore Prometh ION platform.This workflow started with the throat swab of the COVID-19 patient,combined reverse transcript PCR,and multi-amplification in one-step to shorten the experiment time,then can quickly and steadily obtain high-quality SARS-Co V-2 genome within 24 h.A comprehensive evaluation of the method was conducted in 42 samples:the sequencing quality of the method was correlated well with the viral load of the samples;high-quality SARS-Co V-2 genome could be obtained stably in the samples with Ct value up to 39.14;data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20;variation analysis indicated that the method can detect the existing and emerging genomic mutations as well;Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing,making it as low as 0.4/10,000 bp.In summary,high-quality SARS-Co V-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARSCo V-2.展开更多
Binary sequences constructed by Legendre symbols are widely used in communication and cryptography since they have many good pseudo-random properties.In this paper,we determine the 2-adic complexity of the sum sequenc...Binary sequences constructed by Legendre symbols are widely used in communication and cryptography since they have many good pseudo-random properties.In this paper,we determine the 2-adic complexity of the sum sequence of any k many Legendre sequences and show that the 2-adic complexity of the sum sequences of any k many Legendre sequences reaches the maximum by proving the case of k=2 and 3,which implies that the sum sequences can resist the attack of rational approximation algorithm.展开更多
murine genomic DNA was surveyed by polymerase chain reaction (PCR) to detect homeobox sequence of mammal. The PCR product (413 bp) was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence ...murine genomic DNA was surveyed by polymerase chain reaction (PCR) to detect homeobox sequence of mammal. The PCR product (413 bp) was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence of a homeobox, which was isolated in this study, designated Gtx 2 were obtained. The result of sequence analysis showed that there is an intron between the nucleotides at position 138 and 139 in the Gtx 2 homeobox. Southern blot analysis revealed that Gtx 2 is likely to exist as a single copy in the murine genome. Comparison of the encoded polypeptide sequence with other homeodomains reveals that Gtx 2 has 96% and 58% identity to that of Gtx 1 and Tcl 3 , respectively.展开更多
Objective:To surveill emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to examine the association with the sample characteristics,and vaccination status.Met...Objective:To surveill emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to examine the association with the sample characteristics,and vaccination status.Methods:The study analyzed 207 RNA positive swab samples received to sequence laboratory during different waves.The N gene cut-off threshold of less than 30 was considered as the major inclusion criteria.Viral RNA was extracted,and elutes were subjected to nanopore sequencing.All the sequencing data were uploaded in the publicly accessible database,GISAID.Results:The Omicron,Delta and Alpha variants accounted for 58%,22%and 4%of the variants throughout the period.Less than 1%were Kappa variant and 16%of the study samples remained unassigned.Omicron variant was circulated among all age groups and in all the provinces.Ct value and variants assigned percentage was 100%in Ct values of 10-15 while only 45%assigned Ct value over 25.Conclusions:The present study examined the emergence,prevalence,and distribution of SARS-CoV-2 variants locally and has shown that nanopore technology-based genome sequencing enables whole genome sequencing in a low resource setting country.展开更多
The second internal transcribed spacer (ITS-2) of Leucocytozoon caulleryi was amplified by PCR using a pair of conserved primers and cloned into the T-T windows of plasmid pGEM-T easy vector. The inserts were successf...The second internal transcribed spacer (ITS-2) of Leucocytozoon caulleryi was amplified by PCR using a pair of conserved primers and cloned into the T-T windows of plasmid pGEM-T easy vector. The inserts were successfully sequenced and the results revealed that the ITS-2 plus flanking sequence (ITS2+) was composed of 270 nucleotides. and the ITS-2 was 113 bp in length. The ITS-2 of L. caulleryi was analysed by NCBI Blast, and the degree of homology of the ITS-2 of L. caulleryi was compared with that of Eimeria tenella , Candida tropicalis and Saccharomyces kluyveri and others by wDNASIS. The results showed that the ITS-2 of L. caulleryi is characteristic and has the greatest similarity with E. tenella (21.2%).展开更多
Objective To establish and validate a novel diabetic retinopathy(DR)risk-prediction model using a whole-exome sequencing(WES)-based machine learning(ML)method.Methods WES was performed to identify potential single nuc...Objective To establish and validate a novel diabetic retinopathy(DR)risk-prediction model using a whole-exome sequencing(WES)-based machine learning(ML)method.Methods WES was performed to identify potential single nucleotide polymorphism(SNP)or mutation sites in a DR pedigree comprising 10 members.A prediction model was established and validated in a cohort of 420 type 2 diabetic patients based on both genetic and demographic features.The contribution of each feature was assessed using Shapley Additive explanation analysis.The efficacies of the models with and without SNP were compared.Results WES revealed that seven SNPs/mutations(rs116911833 in TRIM7,1997T>C in LRBA,1643T>C in PRMT10,rs117858678 in C9orf152,rs201922794 in CLDN25,rs146694895 in SH3GLB2,and rs201407189 in FANCC)were associated with DR.Notably,the model including rs146694895 and rs201407189 achieved better performance in predicting DR(accuracy:80.2%;sensitivity:83.3%;specificity:76.7%;area under the receiver operating characteristic curve[AUC]:80.0%)than the model without these SNPs(accuracy:79.4%;sensitivity:80.3%;specificity:78.3%;AUC:79.3%).Conclusion Novel SNP sites associated with DR were identified in the DR pedigree.Inclusion of rs146694895 and rs201407189 significantly enhanced the performance of the ML-based DR prediction model.展开更多
This paper deals with part sequencing and optimal robot moves sequence in 2-machine robotic cells according to Petri net graph. We have assumed that the robotic cell is capable of producing same and different parts. W...This paper deals with part sequencing and optimal robot moves sequence in 2-machine robotic cells according to Petri net graph. We have assumed that the robotic cell is capable of producing same and different parts. We have considered a new motion cycle for robot moves sequence which is the development of existing motion cycles in 2-machine robotic cells. The main goal of this study is to minimize the cycle time by determining the optimal part sequencing and robot moves sequence in the robotic cell. So, we have proposed a model based on Petri network.展开更多
Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the...Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.展开更多
Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvari...Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The European Centre for Disease Prevention and Control (eCDC) and the World Health Organization (WHO) recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the polymerase chain reaction (PCR)-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490 - 509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariants when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.2 RBD and an atypical BA.1 NTD, which can only be detected by using specially designed PCR primers. In addition, Sanger sequencing may reveal unclassified subvariants, such as BA.4/BA.5 with L84I mutation in the S gene NTD. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.展开更多
Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in...Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.展开更多
Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylog...Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.展开更多
[Objective]The paper was to understand the polymorphism of TLR2 gene in native Hainan pig breeds.[Method]TLR2 gene were cloned from blood samples of Wuzhishan pigs,Lingao pigs and Tunchang pigs by PCR and sequenced.[R...[Objective]The paper was to understand the polymorphism of TLR2 gene in native Hainan pig breeds.[Method]TLR2 gene were cloned from blood samples of Wuzhishan pigs,Lingao pigs and Tunchang pigs by PCR and sequenced.[Result]The DNA sequence of TLR2 gene in native Hainan pig breeds was 2649 bp and its CDS was 2358 bp.The intra-specific alignment of TLR2 gene sequences of the three pigs showed that there were seven nucleotide polymorphisms in Wuzhishan pigs(two of which located in the coding region),five nucleotide polymorphisms in Tunchang pigs(all of which were outside the coding region)and four nucleotide polymorphisms in Lingao pigs(one of which was located in the coding region).The results of inter-specific comparison showed that there were 12 nucleotide polymorphisms in three pig breeds,three of polymorphisms were missense mutations,resulting in changes in amino acids.The results of homologous analysis showed that the TLR2 gene sequences of the three pig breeds were highly conservative,with the homology ranging from 99.6% to 99.9%.Prediction and analysis of protein structure showed that AG mutation at 876 and 1454 sites of TLR2 gene in native Hainan pigs caused changes in secondary and tertiary structure of the protein,suggesting there might be possible changes in the function of TLR2.[Conclusion]The study provided reference for further research on the relationship between polymorphism of TLR2 gene and epidemic susceptibility of pigs.展开更多
Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation...Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.展开更多
基金Supported by a Sub-project of 973 Program of China(2005CB523001)~~
文摘[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.
基金Supported by D.O.Ott Research Institute of Obstetrics,Gynaecology and Reproductology,project 558-2019-0012(АААА-А19-119021290033-1)of FSBSI
文摘Type 2 diabetes(T2D)mellitus is a common complex disease that currently affects more than 400 million people worldwide and has become a global health problem.High-throughput sequencing technologies such as whole-genome and whole-exome sequencing approaches have provided numerous new insights into the molecular bases of T2D.Recent advances in the application of sequencing technologies to T2D research include,but are not limited to:(1)Fine mapping of causal rare and common genetic variants;(2)Identification of confident genelevel associations;(3)Identification of novel candidate genes by specific scoring approaches;(4)Interrogation of disease-relevant genes and pathways by transcriptional profiling and epigenome mapping techniques;and(5)Investigation of microbial community alterations in patients with T2D.In this work we review these advances in application of next-generation sequencing methods for elucidation of T2D pathogenesis,as well as progress and challenges in implementation of this new knowledge about T2D genetics in diagnosis,prevention,and treatment of the disease.
基金Supported by China Postdoctoral Science Foundation Funded Project(No.2017M612211)Shandong Provincial Natural Science Foundation(No.ZR2018MH016)+3 种基金Qingdao Postdoctoral Application Research Project(No.40518060071)Medical Program of Shandong Province(No.2016WS0265)Qingdao Science and Technology Plan(No.16-6-2-14-nsh)Shandong Province Higher Educational Science and Technology Program(No.J17KA235)
文摘AIM：To summarize the phenotypes and identify the underlying genetic cause of the CRYBB1 and CRYBB2 gene responsible for congenital cataract in two Chinese families.METHODS：Detailed family histories and clinical data were collected from patients during an ophthalmologic examination. Of 523 inheritable genetic vision systemrelated genes were captured and sequenced by targeted next-generation sequencing,and the results were confirmed by Sanger sequencing. The possible functional impacts of an amino acid substitution were performed with Poly Phen-2 and SIFT predictions.RESULTS：The patients in the two families were affected with congenital cataract. Sixty-five （FAMILY-1） and sixty two （FAMILY-2） single-nucleotide polymorphisms and indels were selected by recommended filtering criteria.Segregation was then analyzed by applying Sanger sequencing with the family members. A heterozygous CRYBB1 mutation in exon 4 （c.347T〉C, p.L116P） was identified in sixteen patients in FAMILY-1. A heterozygous CRYBB2 mutation in exon 5 （c.355G〉A, p.G119R） was identified in three patients in FAMILY-2. Each mutation cosegregated with the affected individuals and did not exist in unaffected family members and 200 unrelated normal controls.The mutation was predicted to be highly conservative and to be deleterious by both PolyPhen-2 and SIFT.CONCLUSION：TheCRYBB1 mutation（c.347T〉C）and CRYBB2 mutation （c.355G〉A） are novel in patients with congenital cataract. We summarize the variable phenotypes among the patients, which expanded the phenotypic spectrum of congenital cataract in a different ethnic background.
基金supported by grants from the Foundation for National Mega Project on Major Infectious Disease Prevention(grant number 2017ZX10103005-005)National Key Research and Development Program of China(2020YFC0845800 and 2020YFC0845600)the National Natural Science Foundation of China(31970548 and 91631110)。
文摘Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing.While the rapid acquisition of SARS-Co V-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background.To address this issue,we modified and evaluated an approach by utilizing SARS-Co V-2-specific amplicon amplification and Oxford Nanopore Prometh ION platform.This workflow started with the throat swab of the COVID-19 patient,combined reverse transcript PCR,and multi-amplification in one-step to shorten the experiment time,then can quickly and steadily obtain high-quality SARS-Co V-2 genome within 24 h.A comprehensive evaluation of the method was conducted in 42 samples:the sequencing quality of the method was correlated well with the viral load of the samples;high-quality SARS-Co V-2 genome could be obtained stably in the samples with Ct value up to 39.14;data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20;variation analysis indicated that the method can detect the existing and emerging genomic mutations as well;Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing,making it as low as 0.4/10,000 bp.In summary,high-quality SARS-Co V-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARSCo V-2.
文摘Binary sequences constructed by Legendre symbols are widely used in communication and cryptography since they have many good pseudo-random properties.In this paper,we determine the 2-adic complexity of the sum sequence of any k many Legendre sequences and show that the 2-adic complexity of the sum sequences of any k many Legendre sequences reaches the maximum by proving the case of k=2 and 3,which implies that the sum sequences can resist the attack of rational approximation algorithm.
文摘murine genomic DNA was surveyed by polymerase chain reaction (PCR) to detect homeobox sequence of mammal. The PCR product (413 bp) was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence of a homeobox, which was isolated in this study, designated Gtx 2 were obtained. The result of sequence analysis showed that there is an intron between the nucleotides at position 138 and 139 in the Gtx 2 homeobox. Southern blot analysis revealed that Gtx 2 is likely to exist as a single copy in the murine genome. Comparison of the encoded polypeptide sequence with other homeodomains reveals that Gtx 2 has 96% and 58% identity to that of Gtx 1 and Tcl 3 , respectively.
文摘Objective:To surveill emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to examine the association with the sample characteristics,and vaccination status.Methods:The study analyzed 207 RNA positive swab samples received to sequence laboratory during different waves.The N gene cut-off threshold of less than 30 was considered as the major inclusion criteria.Viral RNA was extracted,and elutes were subjected to nanopore sequencing.All the sequencing data were uploaded in the publicly accessible database,GISAID.Results:The Omicron,Delta and Alpha variants accounted for 58%,22%and 4%of the variants throughout the period.Less than 1%were Kappa variant and 16%of the study samples remained unassigned.Omicron variant was circulated among all age groups and in all the provinces.Ct value and variants assigned percentage was 100%in Ct values of 10-15 while only 45%assigned Ct value over 25.Conclusions:The present study examined the emergence,prevalence,and distribution of SARS-CoV-2 variants locally and has shown that nanopore technology-based genome sequencing enables whole genome sequencing in a low resource setting country.
基金supported by grants from National Natural Science Foundation of China(39870549)Natural Science Foundation of Guangdong Province(980134).
文摘The second internal transcribed spacer (ITS-2) of Leucocytozoon caulleryi was amplified by PCR using a pair of conserved primers and cloned into the T-T windows of plasmid pGEM-T easy vector. The inserts were successfully sequenced and the results revealed that the ITS-2 plus flanking sequence (ITS2+) was composed of 270 nucleotides. and the ITS-2 was 113 bp in length. The ITS-2 of L. caulleryi was analysed by NCBI Blast, and the degree of homology of the ITS-2 of L. caulleryi was compared with that of Eimeria tenella , Candida tropicalis and Saccharomyces kluyveri and others by wDNASIS. The results showed that the ITS-2 of L. caulleryi is characteristic and has the greatest similarity with E. tenella (21.2%).
基金supported by the National Natural Science Foundation of China[Grant No.62206185]。
文摘Objective To establish and validate a novel diabetic retinopathy(DR)risk-prediction model using a whole-exome sequencing(WES)-based machine learning(ML)method.Methods WES was performed to identify potential single nucleotide polymorphism(SNP)or mutation sites in a DR pedigree comprising 10 members.A prediction model was established and validated in a cohort of 420 type 2 diabetic patients based on both genetic and demographic features.The contribution of each feature was assessed using Shapley Additive explanation analysis.The efficacies of the models with and without SNP were compared.Results WES revealed that seven SNPs/mutations(rs116911833 in TRIM7,1997T>C in LRBA,1643T>C in PRMT10,rs117858678 in C9orf152,rs201922794 in CLDN25,rs146694895 in SH3GLB2,and rs201407189 in FANCC)were associated with DR.Notably,the model including rs146694895 and rs201407189 achieved better performance in predicting DR(accuracy:80.2%;sensitivity:83.3%;specificity:76.7%;area under the receiver operating characteristic curve[AUC]:80.0%)than the model without these SNPs(accuracy:79.4%;sensitivity:80.3%;specificity:78.3%;AUC:79.3%).Conclusion Novel SNP sites associated with DR were identified in the DR pedigree.Inclusion of rs146694895 and rs201407189 significantly enhanced the performance of the ML-based DR prediction model.
文摘This paper deals with part sequencing and optimal robot moves sequence in 2-machine robotic cells according to Petri net graph. We have assumed that the robotic cell is capable of producing same and different parts. We have considered a new motion cycle for robot moves sequence which is the development of existing motion cycles in 2-machine robotic cells. The main goal of this study is to minimize the cycle time by determining the optimal part sequencing and robot moves sequence in the robotic cell. So, we have proposed a model based on Petri network.
文摘Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.
文摘Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The European Centre for Disease Prevention and Control (eCDC) and the World Health Organization (WHO) recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the polymerase chain reaction (PCR)-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490 - 509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariants when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.2 RBD and an atypical BA.1 NTD, which can only be detected by using specially designed PCR primers. In addition, Sanger sequencing may reveal unclassified subvariants, such as BA.4/BA.5 with L84I mutation in the S gene NTD. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.
基金supported by the Jiangsu Provincial Key Research and Development Program (Grant No.BE2020616)the National Key R&D Program of China (Grant No.2018YFC1200603)+1 种基金the National Science and Technology Major Project (Grant No.2019SWAQ05-5-4)Jiangsu Key Lab of Cancer Biomarkers,Prevention and Treatment,Collaborative Innovation Center for Cancer Personalized Medicine,Nanjing Medical University.
文摘Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.
基金supported by National Bai Qian Wan Talents Engineering Foudation (Grant No. 9452006-03 )Guangxi Science Technology Bureau (GKG- 0719004-3A)Guangxi Husbandry and Fisheries Bureau.
文摘Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.
基金Supported by Innovation Research Team Project of Natural Science Foundation of Hainan Province(2018CXTD345)Agricultural Science and Technology Innovation Project of Hainan Academy of Agricultural Sciences in 2019"Immunological Enhancement Effect of Traditional Chinese Medicine on Swine Mycoplasma Pneumonia Vaccine"+2 种基金Regional Science Foundation Program of National Natural Science Foundation of China(31560696)Agricultural Science and Technology Innovation Project of Hainan Academy of Agricultural Sciences(CXZX201504)Special Funds for Central Government Guiding Local Science and Technology Development(ZY2019HN01)。
文摘[Objective]The paper was to understand the polymorphism of TLR2 gene in native Hainan pig breeds.[Method]TLR2 gene were cloned from blood samples of Wuzhishan pigs,Lingao pigs and Tunchang pigs by PCR and sequenced.[Result]The DNA sequence of TLR2 gene in native Hainan pig breeds was 2649 bp and its CDS was 2358 bp.The intra-specific alignment of TLR2 gene sequences of the three pigs showed that there were seven nucleotide polymorphisms in Wuzhishan pigs(two of which located in the coding region),five nucleotide polymorphisms in Tunchang pigs(all of which were outside the coding region)and four nucleotide polymorphisms in Lingao pigs(one of which was located in the coding region).The results of inter-specific comparison showed that there were 12 nucleotide polymorphisms in three pig breeds,three of polymorphisms were missense mutations,resulting in changes in amino acids.The results of homologous analysis showed that the TLR2 gene sequences of the three pig breeds were highly conservative,with the homology ranging from 99.6% to 99.9%.Prediction and analysis of protein structure showed that AG mutation at 876 and 1454 sites of TLR2 gene in native Hainan pigs caused changes in secondary and tertiary structure of the protein,suggesting there might be possible changes in the function of TLR2.[Conclusion]The study provided reference for further research on the relationship between polymorphism of TLR2 gene and epidemic susceptibility of pigs.
文摘Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.
