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
Detecting exogenous gene copy numbers is crucial for examining genetically modified organisms. Traditional methods face a trade-off between feasibility and cost, highlighting the need for a high-precision and low-cost...Detecting exogenous gene copy numbers is crucial for examining genetically modified organisms. Traditional methods face a trade-off between feasibility and cost, highlighting the need for a high-precision and low-cost alternative. In this study, we introduced the RISE method, which is based on Rare Allele Infusion and Sanger Sequencing Estimation, for rice.展开更多
With the wide application of DNA sequencing technology, DNA sequences are still increasingly generated through the Sanger sequencing platform. SeqMan （in the LaserGene package） is an excellent program with an easy-t...With the wide application of DNA sequencing technology, DNA sequences are still increasingly generated through the Sanger sequencing platform. SeqMan （in the LaserGene package） is an excellent program with an easy-to-use graphical user interface （GUI） employed to assemble Sanger sequences into contigs. However, with increasing data size, larger sample sets and more sequenced loci make contig assemble complicated due to the considerable number of manual operations required to run SeqMan. Here, we present the ＇autoSeqMan＇ software program, which can automatedly assemble contigs using SeqMan scripting language. There are two main modules available, namely, ＇Classification＇ and ＇Assembly＇. Classification first undertakes preprocessing work, whereas Assembly generates a SeqMan script to consecutively assemble contigs for the classified files. Through comparison with manual operation, we showed that autoSeqMan saved substantial time in the preprocessing and assembly of Sanger sequences. We hope this tool will be useful for those with large sample sets to analyze, but with little programming experience. It is freely available at https：//github.com/ Sun-Yanbo/autoSeqMan.展开更多
The seeds for genomics were sown with the development of DNA sequencing(Sanger et al., 1977), cultivated with each advance in molecular biology, and have since grown into one of the most important aspects of the life ...The seeds for genomics were sown with the development of DNA sequencing(Sanger et al., 1977), cultivated with each advance in molecular biology, and have since grown into one of the most important aspects of the life sciences. With the sequencing of the first draft human genome(Venter et al.,2001), the era of post-genomics began. Once one genome has been sequenced, it can be used as the reference for a certain species, and sequences from individuals can be mapped onto it to compare genetic variations among different lines.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
Background: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and eva...Background: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA. Methods: A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, turnaround time and cost of these two strategies were compared. Results: Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The turnaround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy. Conclusion: Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.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
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
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
The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specif...The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specificity and sensitivity are highly variable, and there is no direct relationship between the level increase of these biomarkers for mycosis. It is common to obtain negative microbiological cultures in patients infected by non-culturable, intracellular bacteria or mycosis, even though there is a high clinical suspicion of infection. This study identifies the pathogen present in critically infected patients through 16S and 18S/eEF1 genes detection by polymerase chain reaction (PCR) coupled with Sanger sequencing. Thirty clinical samples were evaluated by PCR, of which 40% were positive for fungi, 23.33% for bacteria, 26.7% for fungi and bacteria, and 10% for no pathogen. The PCRs outcomes period for bacteria or fungi was one day compared to seven and up to 14 days (on average) of microbiological culture for bacteria and fungi. Then, we assessed the relationship with the most used biomarkers (procalcitonin, C-reactive protein, globular sedimentation velocity, and the neutrophil-lymphocyte index). This combination of molecular techniques has been shown as helpful in identifying intracellular bacteria and fungi that are difficult to culture by conventional methods. Screening with genomic markers 16S and 18S/eEF1 by PCR allowed us to optimize the time to obtain the result of the infection caused by bacteria or fungi. Also, identifying the specific etiological microorganism by Sanger sequencing was very helpful in avoiding the progression of the disease and setting targeted treatment with better clinical outcomes.展开更多
background Mendelian stroke causes nearly 7% of ischaemic strokes and is also an important aetiology of cryptogenic stroke.Identifying the genetic abnormalities in Mendelian strokes is important as it would facilitate...background Mendelian stroke causes nearly 7% of ischaemic strokes and is also an important aetiology of cryptogenic stroke.Identifying the genetic abnormalities in Mendelian strokes is important as it would facilitate therapeutic management and genetic counselling.Next-generation sequencing makes large-scale sequencing and genetic testing possible.Methods A systematic literature search was conducted to identify causal genes of Mendelian strokes,which were used to construct a hybridization-based gene capture panel.Genetic variants for target genes were detected using Illumina HiSeq X10 and the Novaseq platform.The sensitivity and specificity were evaluated by comparing the results with Sanger sequencing.results 53 suspected patients of Mendelian strokes were analysed using the panel of 181 causal genes.According to the American College of Medical Genetics and Genomics standard,16 likely pathogenic/variants of uncertain significance genetic variants were identified.Diagnostic testing was conducted by comparing the consistency between the results of panel and Sanger sequencing.Both the sensitivity and specificity were 100%for the panel.Conclusion This panel provides an economical,time-saving and labour-saving method to detect causal mutations of Mendelian strokes.展开更多
The CRISPR(clustered regularly interspaced short palindromic repeats)-associated protein 9(Cas9)system is a powerful tool for targeted genome editing,with applications that include plant biotechnology and functional g...The CRISPR(clustered regularly interspaced short palindromic repeats)-associated protein 9(Cas9)system is a powerful tool for targeted genome editing,with applications that include plant biotechnology and functional genomics research.However,the specificity of Cas9 targeting is poorly investigated in many plant species,including fruit trees.To assess the off-target mutation rate in grapevine(Vitis vinifera),we performed whole-genome sequencing(WGS)of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type(WT)plants.In total,we identified between 202,008 and 272,397 single nucleotide polymorphisms(SNPs)and between 26,391 and 55,414 insertions/deletions(indels)in the seven Cas9-edited grapevine plants compared with the three WT plants.Subsequently,3272 potential off-target sites were selected for further analysis.Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing.In addition,we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome(PN40024)but no true off-target mutations.In conclusion,we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.展开更多
Few studies have explored the differences between Sanger and HTS methods in the results of mitogenome sequencing. We used a single individual of insect to study the differences between the sequences given by Sanger an...Few studies have explored the differences between Sanger and HTS methods in the results of mitogenome sequencing. We used a single individual of insect to study the differences between the sequences given by Sanger and PCR-free HTS methods. Here we provided evidence for biased results of sequencing due to different methods in the mitochondrial genes of atp6, atp8, cox1, cox2, cox3, Cytb, nad2, nad3, nad4, nad5, rrn S, rrnL, trnH, trn I, and control region at various degrees. Especially, in cox1, the differently sequenced nucleotides account for 2.6% of the complete length. Furthermore, the highest value of the intraspecific genetic distance based on K2 P accounts for 2.5% using a barcode fragment size of cox1（651 bp, Sanger）, while the maximum distance of the corresponding cox1 fragment obtained by the two sequencing methods was 5.0%. We revealed that the methods of Sanger and HTS may give different sequencing results of mitochondrial genes, which may reflect the heteroplasmy of mitogenomes within an insect individual. Therefore, researchers should be very cautious in using the mixed data of a gene given by different methods of sequencing.展开更多
Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites i...Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites is cost-and time-intensive. Orchidaceae is one of the most species-rich families of angiosperms with more than 30,000 species estimated. Despite its high species-diversity, microsatellites are available only for a few species and all were developed by only using Sanger sequencing methods. For the first time in orchids, we used 454 GS-FLX sequencing to isolate microsatellites in two species (Cypripedium kentuckiense and Pogonia ophioglossoides), and report preliminary results of the study. From 1/16th plate that was subjected to sequencing, 32,665 reads were generated, from which 15,473 fragments contained at least one SSR. We selected 20,697 SSRs representing di-, tri-, and tetra-nucleotides. While 3,674 microsatellites had flanking regions on both sides, useable primer pairs could be designed for 255 SSRs. The mean numbers of reads, SSRs, and SSR-containing reads useful for primer design estimated for other 15 orchid species using Sanger sequencing method were 166, 78 and 31, respectively. Results demonstrate that the efficiency of microsatellite isolation in orchids is substantially higher with 454 GS-FLX sequencing technique in comparison to the Sanger sequencing methods.展开更多
AIMTo identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD).METHODSA southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-...AIMTo identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD).METHODSA southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-exome sequencing (WES), coupling the Agilent whole-exome capture system to the Illumina HiSeq 2000 DNA sequencing platform was used to search the specific gene mutation in 3 affected family members and 1 unaffected member. After a suggested variant was found through the data analysis, the putative mutation was validated by Sanger DNA sequencing of samples from all available family members.RESULTSThe results of both WES and Sanger sequencing revealed a novel nonsense mutation c.C766T (p.Q256X) within exon 5 of CRX gene which was pathogenic for adCORD in this family. The mutation could affect photoreceptor-specific gene expression with a dominant-negative effect and resulted in loss of the OTX tail, thus the mutant protein occupies the CRX-binding site in target promoters without establishing an interaction and, consequently, may block transactivation.CONCLUSIONAll modes of Mendelian inheritance in CORD have been observed, and genetic heterogeneity is a hallmark of CORD. Therefore, conventional genetic diagnosis of CORD would be time-consuming and labor-intensive. Our study indicated the robustness and cost-effectiveness of WES in the genetic diagnosis of CORD.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.
基金supported by the Natural Science Foundation of Hunan Province for Distinguished Young Scholars, China (Grant No.2021JJ10041)the Biological Breeding-National Science and Technology Major Project, China (Grant No.2023ZD04072)the Hainan Yazhou Bay Seed Laboratory and National Seed Group, China (Grant Nos.B23CQ15HP and B23YQ1517)。
文摘Detecting exogenous gene copy numbers is crucial for examining genetically modified organisms. Traditional methods face a trade-off between feasibility and cost, highlighting the need for a high-precision and low-cost alternative. In this study, we introduced the RISE method, which is based on Rare Allele Infusion and Sanger Sequencing Estimation, for rice.
基金supported by the National Natural Science Foundation of China(31671326)the Youth Innovation Promotion Association,Chinese Academy of Sciences
文摘With the wide application of DNA sequencing technology, DNA sequences are still increasingly generated through the Sanger sequencing platform. SeqMan （in the LaserGene package） is an excellent program with an easy-to-use graphical user interface （GUI） employed to assemble Sanger sequences into contigs. However, with increasing data size, larger sample sets and more sequenced loci make contig assemble complicated due to the considerable number of manual operations required to run SeqMan. Here, we present the ＇autoSeqMan＇ software program, which can automatedly assemble contigs using SeqMan scripting language. There are two main modules available, namely, ＇Classification＇ and ＇Assembly＇. Classification first undertakes preprocessing work, whereas Assembly generates a SeqMan script to consecutively assemble contigs for the classified files. Through comparison with manual operation, we showed that autoSeqMan saved substantial time in the preprocessing and assembly of Sanger sequences. We hope this tool will be useful for those with large sample sets to analyze, but with little programming experience. It is freely available at https：//github.com/ Sun-Yanbo/autoSeqMan.
基金supported by the Ministry of Agriculture of China(2016ZX08009-003)the Chinese Academy of Sciences(ZDRWZS-2019-2)the National Natural Science Foundation of China(31788103)。
文摘The seeds for genomics were sown with the development of DNA sequencing(Sanger et al., 1977), cultivated with each advance in molecular biology, and have since grown into one of the most important aspects of the life sciences. With the sequencing of the first draft human genome(Venter et al.,2001), the era of post-genomics began. Once one genome has been sequenced, it can be used as the reference for a certain species, and sequences from individuals can be mapped onto it to compare genetic variations among different lines.
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.
基金grants from The National Key Research and Development Program of China (No.2016YFC0901505)National Natural Science Foundation of China (No.81500979)+3 种基金CAMS Initiative for Innovative Medicine (CAMS-I2M-1-008)Central Research Institutes of Basic Research and Public Service Special Operations (No.2016ZX310182-6)a SpecialFund for Capital Health Research and Development (No.2011-1008-03)the Natural Science Foundation of Beijing Municipality (No.5163028).
文摘Background: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA. Methods: A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, turnaround time and cost of these two strategies were compared. Results: Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The turnaround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy. Conclusion: Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.
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.
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.
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.
文摘The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specificity and sensitivity are highly variable, and there is no direct relationship between the level increase of these biomarkers for mycosis. It is common to obtain negative microbiological cultures in patients infected by non-culturable, intracellular bacteria or mycosis, even though there is a high clinical suspicion of infection. This study identifies the pathogen present in critically infected patients through 16S and 18S/eEF1 genes detection by polymerase chain reaction (PCR) coupled with Sanger sequencing. Thirty clinical samples were evaluated by PCR, of which 40% were positive for fungi, 23.33% for bacteria, 26.7% for fungi and bacteria, and 10% for no pathogen. The PCRs outcomes period for bacteria or fungi was one day compared to seven and up to 14 days (on average) of microbiological culture for bacteria and fungi. Then, we assessed the relationship with the most used biomarkers (procalcitonin, C-reactive protein, globular sedimentation velocity, and the neutrophil-lymphocyte index). This combination of molecular techniques has been shown as helpful in identifying intracellular bacteria and fungi that are difficult to culture by conventional methods. Screening with genomic markers 16S and 18S/eEF1 by PCR allowed us to optimize the time to obtain the result of the infection caused by bacteria or fungi. Also, identifying the specific etiological microorganism by Sanger sequencing was very helpful in avoiding the progression of the disease and setting targeted treatment with better clinical outcomes.
基金The Ministry of Science and Technology of the People’s Republic of China(2016YFC0901001,2016YFC0901002,2016YFC0901004,2017YFC1310901,2017YFC1310902,2018YFC1311700,2018YFC1311706)National Science and Technology Major Project(2017ZX09304018)+1 种基金Beijing Municipal Commission of Health and Family Planning(No.2016-1-2041,SML20150502)Beijing Municipal Science&Technology Commission(D171100003017002).
文摘background Mendelian stroke causes nearly 7% of ischaemic strokes and is also an important aetiology of cryptogenic stroke.Identifying the genetic abnormalities in Mendelian strokes is important as it would facilitate therapeutic management and genetic counselling.Next-generation sequencing makes large-scale sequencing and genetic testing possible.Methods A systematic literature search was conducted to identify causal genes of Mendelian strokes,which were used to construct a hybridization-based gene capture panel.Genetic variants for target genes were detected using Illumina HiSeq X10 and the Novaseq platform.The sensitivity and specificity were evaluated by comparing the results with Sanger sequencing.results 53 suspected patients of Mendelian strokes were analysed using the panel of 181 causal genes.According to the American College of Medical Genetics and Genomics standard,16 likely pathogenic/variants of uncertain significance genetic variants were identified.Diagnostic testing was conducted by comparing the consistency between the results of panel and Sanger sequencing.Both the sensitivity and specificity were 100%for the panel.Conclusion This panel provides an economical,time-saving and labour-saving method to detect causal mutations of Mendelian strokes.
基金the National Natural Science Foundation of China(U1603234,31572110,and 32002000)the Program for Innovative Research Team of Grape Germplasm Resources and Breeding(2013KCT-25)。
文摘The CRISPR(clustered regularly interspaced short palindromic repeats)-associated protein 9(Cas9)system is a powerful tool for targeted genome editing,with applications that include plant biotechnology and functional genomics research.However,the specificity of Cas9 targeting is poorly investigated in many plant species,including fruit trees.To assess the off-target mutation rate in grapevine(Vitis vinifera),we performed whole-genome sequencing(WGS)of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type(WT)plants.In total,we identified between 202,008 and 272,397 single nucleotide polymorphisms(SNPs)and between 26,391 and 55,414 insertions/deletions(indels)in the seven Cas9-edited grapevine plants compared with the three WT plants.Subsequently,3272 potential off-target sites were selected for further analysis.Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing.In addition,we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome(PN40024)but no true off-target mutations.In conclusion,we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.
基金supported by the National Natural Science Foundation of China(31222051)
文摘Few studies have explored the differences between Sanger and HTS methods in the results of mitogenome sequencing. We used a single individual of insect to study the differences between the sequences given by Sanger and PCR-free HTS methods. Here we provided evidence for biased results of sequencing due to different methods in the mitochondrial genes of atp6, atp8, cox1, cox2, cox3, Cytb, nad2, nad3, nad4, nad5, rrn S, rrnL, trnH, trn I, and control region at various degrees. Especially, in cox1, the differently sequenced nucleotides account for 2.6% of the complete length. Furthermore, the highest value of the intraspecific genetic distance based on K2 P accounts for 2.5% using a barcode fragment size of cox1（651 bp, Sanger）, while the maximum distance of the corresponding cox1 fragment obtained by the two sequencing methods was 5.0%. We revealed that the methods of Sanger and HTS may give different sequencing results of mitochondrial genes, which may reflect the heteroplasmy of mitogenomes within an insect individual. Therefore, researchers should be very cautious in using the mixed data of a gene given by different methods of sequencing.
文摘Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites is cost-and time-intensive. Orchidaceae is one of the most species-rich families of angiosperms with more than 30,000 species estimated. Despite its high species-diversity, microsatellites are available only for a few species and all were developed by only using Sanger sequencing methods. For the first time in orchids, we used 454 GS-FLX sequencing to isolate microsatellites in two species (Cypripedium kentuckiense and Pogonia ophioglossoides), and report preliminary results of the study. From 1/16th plate that was subjected to sequencing, 32,665 reads were generated, from which 15,473 fragments contained at least one SSR. We selected 20,697 SSRs representing di-, tri-, and tetra-nucleotides. While 3,674 microsatellites had flanking regions on both sides, useable primer pairs could be designed for 255 SSRs. The mean numbers of reads, SSRs, and SSR-containing reads useful for primer design estimated for other 15 orchid species using Sanger sequencing method were 166, 78 and 31, respectively. Results demonstrate that the efficiency of microsatellite isolation in orchids is substantially higher with 454 GS-FLX sequencing technique in comparison to the Sanger sequencing methods.
基金Supported by the Zhejiang Provincial Natural Science Foundation of China (No.LY12H12001)the Ningbo Key Foundation of Society Development (No.2014C50091)+2 种基金the Ningbo Natural Science Foundation (No.2012A610192)the Ningbo Yinzhou District S&T Foundation (No.YK2013-90)the Shenzhen Municipal Government of China (No.GJHZ20130417140916986)
文摘AIMTo identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD).METHODSA southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-exome sequencing (WES), coupling the Agilent whole-exome capture system to the Illumina HiSeq 2000 DNA sequencing platform was used to search the specific gene mutation in 3 affected family members and 1 unaffected member. After a suggested variant was found through the data analysis, the putative mutation was validated by Sanger DNA sequencing of samples from all available family members.RESULTSThe results of both WES and Sanger sequencing revealed a novel nonsense mutation c.C766T (p.Q256X) within exon 5 of CRX gene which was pathogenic for adCORD in this family. The mutation could affect photoreceptor-specific gene expression with a dominant-negative effect and resulted in loss of the OTX tail, thus the mutant protein occupies the CRX-binding site in target promoters without establishing an interaction and, consequently, may block transactivation.CONCLUSIONAll modes of Mendelian inheritance in CORD have been observed, and genetic heterogeneity is a hallmark of CORD. Therefore, conventional genetic diagnosis of CORD would be time-consuming and labor-intensive. Our study indicated the robustness and cost-effectiveness of WES in the genetic diagnosis of CORD.
