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
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
BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients wi...BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis,which can be very poor.AIM To identify pathogenic genes in DCM through pedigree analysis.METHODS Our research team identified a patient with DCM in the clinic.Through invest-igation,we found that the family of this patient has a typical DCM pedigree.High-throughput sequencing technology,next-generation sequencing,was used to sequence the whole exomes of seven samples in the pedigree.RESULTS A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered.The mutation was completely consistent with the clinical information for this DCM pedigree.Sanger sequencing was used to further verify the locus of the mutation in pedigree samples.These results were consistent with those of high-throughput sequencing.CONCLUSIONS ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.展开更多
The purpose of the present study was to study the characteristics of epidemic growth factor receptor(EGFR)gene distribution in patients with non-small cell lung cancer(NSCLC),and to detect the mutation rate of EGFR ge...The purpose of the present study was to study the characteristics of epidemic growth factor receptor(EGFR)gene distribution in patients with non-small cell lung cancer(NSCLC),and to detect the mutation rate of EGFR gene by Sanger sequencing and amplification refractory mutation system(ARMS)-PCR.Paraffin-embedded sections of NSCLC tissues from 399 NSCLC patients diagnosed in Renmin Hospital of Wuhan University were collected,103 of them were detected for exons 18-21 mutation of EGFR by Sanger sequencing method,296 cases were detected for exons 18-21 mutation by ARMS-PCR method.DNA extraction of both groups was performed with Qiagen QLAamp DNA FFPE Tissue KIT.Comparisons of detection rates between the two methods were conducted by row X list chi-square test.The total mutation rate of EGFR gene detected by Sanger sequencing was 21.4%,exons 18-21 and combined mutation rates were 1.0%,9.7%,1.0%,7.8%and 2.0%,respectively.And the proportions were 4.7%,45.2%,4.7%,36.3%and 9.4%respectively.The total mutation rate detected by ARMS-PCR was 51.4%,exons 18-21 and combined mutation rates were 2.7%,27%,1.7%,18.2%and 1.7%,respectively.The proportions were 5.3%,52.6%,3.3%,35.5%and 3.3%respectively.Further analysis of mutation rate showed that there was significant difference between the two methods in detecting total mutation of EGFR gene(P<0.001).There were significant differences in mutation detection rates of exons 19 and 21(P<0.001,P<0.05),but there were no significant differences in other exons.And there was no significant difference in mutation detection rates between the two methods.The mutation rate of EGFR gene in NSCLC patients was 50%.And exon 19 deletion was the most common mutation type,followed by exon 21 mutation.Compared with Sanger sequencing method,ARMS method is more sensitive with significant advantages in detecting exon 19 deletions and exon 21 mutations,which can be widely used in clinical detection of EGFR gene mutations.The results of this study will further guide patients with advanced NSCLC to select TKI targeted drugs,and provide clinical diagnostic basis for targeted therapy of NSCLC patients.展开更多
Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in deva...Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in devastating damage, causing huge economic losses to its farmers. In present study, molecular typing is performed on basis of partially conserved hexon gene sequences, using a unique set of primers having common reverse oligo for simultaneous detection of FAdV1, FAdV-4 and FAdV-11. A total of 14 fowl adeno virus strains were isolated from 100 suspected adeno virus liver samples, collected from different districts in Pakistan, between 2018 and 2019. FASTA’s sequence alignment and phylogenetic analysis revealed that out of the 14, one isolate which belonged to group A showed 27% similarity with FAdV-1, while three isolates showed 99%, 95% & 45% similarity to FAdV-4 (Group C). Whereas, ten isolates showed more than 99% similarity to FAdV-11 (Group D). The serotypes FAdV1, FAdV-4 and FAdV-11 are prevailing in the breeder and broilers. These results hold great importance in rapid, reliable and simultaneous detection of the three FAdV serotypes. Therefore, fowl adeno virus vaccine production for commercial poultry shall be according to the prevalent field serotypes.展开更多
DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when req...DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing(HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing,HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITACseq was effective in detecting relatively large structural variations, as demonstrated by the identification of a~1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many highthroughput sequencing and genotyping applications.展开更多
Infections by nonpolio enteroviruses(EVs)are highly prevalent,particularly among children and neonates,where they may cause substantial morbidity and mortality.Laboratory diagnosis of these viral infections is importa...Infections by nonpolio enteroviruses(EVs)are highly prevalent,particularly among children and neonates,where they may cause substantial morbidity and mortality.Laboratory diagnosis of these viral infections is important in patient prognosis and guidance of clinical management.Although the laboratory diagnosis of non-polio EVs is mainly based on molecular techniques,classical virus-isolation techniques are still used in refer-ence laboratories.Other techniques,such as antigen detection and serology,are becoming obsolete and rarely used in diagnosis.An important part of diagnosis and surveillance of EV infections is viral typing by VP1 gene sequencing using conventional Sanger technique and more recently,full-genome next-generation sequencing.The latter allows the typing of all EVs,better investigation of EV outbreaks,detection of coinfec-tion,and identification of severity markers in the EV genome.展开更多
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.
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 Jilin Provincial Healthcare Talent Special Program,No.2019SCZT08.
文摘BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis,which can be very poor.AIM To identify pathogenic genes in DCM through pedigree analysis.METHODS Our research team identified a patient with DCM in the clinic.Through invest-igation,we found that the family of this patient has a typical DCM pedigree.High-throughput sequencing technology,next-generation sequencing,was used to sequence the whole exomes of seven samples in the pedigree.RESULTS A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered.The mutation was completely consistent with the clinical information for this DCM pedigree.Sanger sequencing was used to further verify the locus of the mutation in pedigree samples.These results were consistent with those of high-throughput sequencing.CONCLUSIONS ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.
基金grants from the National Natural Science Foundation of China(No.81100959)Natural Science Fund of Hubei Province(No.2015CFB185).
文摘The purpose of the present study was to study the characteristics of epidemic growth factor receptor(EGFR)gene distribution in patients with non-small cell lung cancer(NSCLC),and to detect the mutation rate of EGFR gene by Sanger sequencing and amplification refractory mutation system(ARMS)-PCR.Paraffin-embedded sections of NSCLC tissues from 399 NSCLC patients diagnosed in Renmin Hospital of Wuhan University were collected,103 of them were detected for exons 18-21 mutation of EGFR by Sanger sequencing method,296 cases were detected for exons 18-21 mutation by ARMS-PCR method.DNA extraction of both groups was performed with Qiagen QLAamp DNA FFPE Tissue KIT.Comparisons of detection rates between the two methods were conducted by row X list chi-square test.The total mutation rate of EGFR gene detected by Sanger sequencing was 21.4%,exons 18-21 and combined mutation rates were 1.0%,9.7%,1.0%,7.8%and 2.0%,respectively.And the proportions were 4.7%,45.2%,4.7%,36.3%and 9.4%respectively.The total mutation rate detected by ARMS-PCR was 51.4%,exons 18-21 and combined mutation rates were 2.7%,27%,1.7%,18.2%and 1.7%,respectively.The proportions were 5.3%,52.6%,3.3%,35.5%and 3.3%respectively.Further analysis of mutation rate showed that there was significant difference between the two methods in detecting total mutation of EGFR gene(P<0.001).There were significant differences in mutation detection rates of exons 19 and 21(P<0.001,P<0.05),but there were no significant differences in other exons.And there was no significant difference in mutation detection rates between the two methods.The mutation rate of EGFR gene in NSCLC patients was 50%.And exon 19 deletion was the most common mutation type,followed by exon 21 mutation.Compared with Sanger sequencing method,ARMS method is more sensitive with significant advantages in detecting exon 19 deletions and exon 21 mutations,which can be widely used in clinical detection of EGFR gene mutations.The results of this study will further guide patients with advanced NSCLC to select TKI targeted drugs,and provide clinical diagnostic basis for targeted therapy of NSCLC patients.
文摘Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in devastating damage, causing huge economic losses to its farmers. In present study, molecular typing is performed on basis of partially conserved hexon gene sequences, using a unique set of primers having common reverse oligo for simultaneous detection of FAdV1, FAdV-4 and FAdV-11. A total of 14 fowl adeno virus strains were isolated from 100 suspected adeno virus liver samples, collected from different districts in Pakistan, between 2018 and 2019. FASTA’s sequence alignment and phylogenetic analysis revealed that out of the 14, one isolate which belonged to group A showed 27% similarity with FAdV-1, while three isolates showed 99%, 95% & 45% similarity to FAdV-4 (Group C). Whereas, ten isolates showed more than 99% similarity to FAdV-11 (Group D). The serotypes FAdV1, FAdV-4 and FAdV-11 are prevailing in the breeder and broilers. These results hold great importance in rapid, reliable and simultaneous detection of the three FAdV serotypes. Therefore, fowl adeno virus vaccine production for commercial poultry shall be according to the prevalent field serotypes.
基金supported by grants from National Key Research and Development Program (2016YFD0101803-04)National Natural Science Foundation of China (31421005 and 91935303)。
文摘DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing(HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing,HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITACseq was effective in detecting relatively large structural variations, as demonstrated by the identification of a~1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many highthroughput sequencing and genotyping applications.
基金This study was funded by the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing,grant ID 121041500041−1.
文摘Infections by nonpolio enteroviruses(EVs)are highly prevalent,particularly among children and neonates,where they may cause substantial morbidity and mortality.Laboratory diagnosis of these viral infections is important in patient prognosis and guidance of clinical management.Although the laboratory diagnosis of non-polio EVs is mainly based on molecular techniques,classical virus-isolation techniques are still used in refer-ence laboratories.Other techniques,such as antigen detection and serology,are becoming obsolete and rarely used in diagnosis.An important part of diagnosis and surveillance of EV infections is viral typing by VP1 gene sequencing using conventional Sanger technique and more recently,full-genome next-generation sequencing.The latter allows the typing of all EVs,better investigation of EV outbreaks,detection of coinfec-tion,and identification of severity markers in the EV genome.
