The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary...The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.展开更多
Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laborat...Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.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
Fish, which are vital for both aquatic ecosystem functionality and global food supply, rely heavily on theirgastrointestinal tract (GIT) microbiota for the digestion that underpins their growth and health. Takifugu pu...Fish, which are vital for both aquatic ecosystem functionality and global food supply, rely heavily on theirgastrointestinal tract (GIT) microbiota for the digestion that underpins their growth and health. Takifugu puf-ferfish, which are an example of species evolved through adaptive radiation, possess a GIT that is specialized forantipredator defense and gluttonous feeding behaviors, offering a unique model to explore the effects of GrTcompartmentalization and host genetics on gut microbial communities. Here we compiled 78 full and partial-length 16 S rRNA amplicon datasets across three anteroposteriorly distinct intestinal sites in a cohort of cohab-itating artificial hybrid and purebred Takifugu pufferfishes. Our findings reveal a compositional and functionalbiogeography of pufferfish gut microbiota along the GIT and between host genetics. Additionally, the differentialabundance of specific amplicon sequence variants and their correlation with host genetic backgrounds and in-testinal sections highlight the role of environmental filtering in shaping microbial communities,with certainbacterial taxa exhibiting strong preferences for particular intestinal sites or genetic backgrounds,suggestingpotential localized adaptation or functional specialization. This study enhances our understanding of the intricateinterplay between host genetics, gut anatomy, and microbiota in fish, underscoring the importance of detailedmicrobial profiling in conservation efforts and aquaculture practices, and emphasizing the necessity of integratingfull-length 16 S rRNA sequencing with partial-length datasets to comprehensively understand microbial diversityand function, paving the way for improved fish health management and sustainable aquaculture strategies.展开更多
A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfec-tion ...A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfec-tion with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 amplicon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-liter rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.展开更多
Background:Cryptococcal meningitis is a severe infectious disease associated with high morbidity and mortality.Rapidity and accuracy of diagnosis contribute to better prognosis,but readily available tools,such as micr...Background:Cryptococcal meningitis is a severe infectious disease associated with high morbidity and mortality.Rapidity and accuracy of diagnosis contribute to better prognosis,but readily available tools,such as microscopy,culture,and antigens do not perform well all the time.Our study attempted to diagnose and genotype cryptococcus in the cerebrospinal fluid(CSF)samples from patients with cryptococcal meningitis using the approach of metataxonomics of Internal Transcribed Spacer(ITS)amplicons.Methods:The CSF samples were collected from 11 clinically suspected cryptococcal meningitis patients and four non-infectious controls.Samples were recruited from the First Affiliated Hospital of Fujian Medical University Hospital,Fuzhou Fourth Hospital and the 476th Hospital of Chinese People's Liberation Army from December 2017 to December 2018.ITS1 ribosomal deoxyribonucleic acid(rDNA)genes of 15 whole samples were amplified by universal forward primer ITS1(CTTGGTCATTTAGAGGAAGTAA)and reverse primer ITS2(GCTGCGTTCTTCATCGATGC),sequenced by Illumina MiSeq Benchtop Sequencer.The results were confirmed by sanger sequencing of ITS1 region and partial CAP59 gene of microbial isolates from 11 meningitic samples.Pair-wise comparison between infectious group and control group was conducted through permutational multivariate analysis(PERMANOVA)in R software.Results:The 30,000 to 340,000 high-quality clean reads were obtained from each of the positively stained or cultured CSF samples and 8 to 60 reads from each control.The samples from 11 infected patients yielded detectable cryptococcal-specific ITS1 DNA with top abundance(from 95.90%to 99.97%),followed by many other fungal groups(each<1.41%).ITS genotype was defined in 11 CSF samples,corresponding to ITS type 1,and confirmed by Sanger sequencing.A statistically significant difference(r2=0.65869,P=0.0014)between infectious group and control group was observed.Conclusions:The metataxonomics of ITS amplicons facilitates the diagnosis and genotype of cryptococcus in CSF samples,which may provide a better diagnostic approach of cryptococcal infection.展开更多
The microbiome associated with brown planthopper(BPH)plays an important role in mediating host health and fitness.Characterization of the microbial community and its structure is prerequisite for understanding the int...The microbiome associated with brown planthopper(BPH)plays an important role in mediating host health and fitness.Characterization of the microbial community and its structure is prerequisite for understanding the intricate symbiotic relationships between microbes and host insect.Here,we investigated the bacterial and fungal communities of BPH at different developmental stages using high-throughput amplicon sequencing.Our results revealed that both the bacterial and fungal communities were diverse and dynamic during BPH development.The bacterial communities were generally richer than fungi in each developmental stage.At 97%similarly,19 phyla and 278 genera of bacteria were an-notated,while five fungal phyla comprising 80 genera were assigned.The highest species richness for the bacterial communities was detected in the nymphal stage.The taxonomic diversity of the fungal communities in female adults was generally at a relatively higher level when compared to other developmental stages.The most dominant phylum of bacteria and fungi at each developmental stage all belonged to Proteobacteria and Ascomycota,re-spectively.A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages,while the dominant fun-gal genus Wallemia was more abundant in the nymph and adult stages than in the egg stage.Additionally,the microbial composition differed between male and female adults,suggesting that the microbial communties In BPH were gender-dependent.Uverall,our study enriches our knowledge on the microbial communities associated with BPH and will provide clues to develop potential biocontrol techniques against this rice pest.展开更多
High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of ...High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.展开更多
基金supported by the National Key Research and Development Program of China(2022YFC2303401,2022YFC2304100,2016YFD0500301,2021YFC0863300)the Beijing Science and Technology Plan(Z211100002521017)the National Natural Science Foundation of China(82241080)。
文摘The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.
基金supported by the National Key Research and Development Program(grant number:2022YFC2305304).
文摘Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.
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.
基金supported by the National Natural Science Foundation of China(42376119 to X.J.)the Project for Seed Industry Vitalization of Jiangsu Province(JBGS[2021]133 to Z.Z.)the Natural Science Foundation of Jiangsu Province(BK20210362 to X.J.).
文摘Fish, which are vital for both aquatic ecosystem functionality and global food supply, rely heavily on theirgastrointestinal tract (GIT) microbiota for the digestion that underpins their growth and health. Takifugu puf-ferfish, which are an example of species evolved through adaptive radiation, possess a GIT that is specialized forantipredator defense and gluttonous feeding behaviors, offering a unique model to explore the effects of GrTcompartmentalization and host genetics on gut microbial communities. Here we compiled 78 full and partial-length 16 S rRNA amplicon datasets across three anteroposteriorly distinct intestinal sites in a cohort of cohab-itating artificial hybrid and purebred Takifugu pufferfishes. Our findings reveal a compositional and functionalbiogeography of pufferfish gut microbiota along the GIT and between host genetics. Additionally, the differentialabundance of specific amplicon sequence variants and their correlation with host genetic backgrounds and in-testinal sections highlight the role of environmental filtering in shaping microbial communities,with certainbacterial taxa exhibiting strong preferences for particular intestinal sites or genetic backgrounds,suggestingpotential localized adaptation or functional specialization. This study enhances our understanding of the intricateinterplay between host genetics, gut anatomy, and microbiota in fish, underscoring the importance of detailedmicrobial profiling in conservation efforts and aquaculture practices, and emphasizing the necessity of integratingfull-length 16 S rRNA sequencing with partial-length datasets to comprehensively understand microbial diversityand function, paving the way for improved fish health management and sustainable aquaculture strategies.
文摘目的揭示道地产区宝鸡西山产柴胡Bupleurum chinense植物根际土壤细菌的群落结构和多样性,为解析西山产柴胡的道地性和生态种植提供一定理论基础。方法采用16S rDNA微生物扩增子测序初步确定根际细菌的群落组成,利用稀释分离法对根际土进行分离纯化并提取细菌DNA进行分子鉴定。结果16S rDNA微生物扩增子测序结果显示样品质量总体良好。属水平上丰度前10的分别是盖亚女神菌属Gaiella、未命名蓝细菌属unidentified_Cyanobacteria、海无柄孢囊黏细菌属Haliangium、土壤红杆菌属Solirubrobacter、假节杆菌属Pseudarthrobacter、出芽菌属Gemmata、芽生球菌属Blastococcus、类诺卡氏菌属Nocardioides、鞘氨醇单胞菌属Sphingomonas、芽单苞菌属Gemmatimonas,除土壤红杆菌属外其余均属于有益菌。代谢通路预测结果显示分布于6大类:Cellular Processes、Environmental Information Processes、Genetic information processe、Human disease、Metabolism、Organismal-system。共分离到16株根际细菌,鉴定后发现均属于芽孢杆菌属,可鉴定到种水平的有漳州芽孢杆菌、短小芽孢杆菌、沙福芽孢杆菌和嗜气芽胞杆菌4种。结论阐明了西山产柴胡根际细菌群落的组成情况,物种丰富度较高且个体分布也相对均匀。在属水平上丰度排名前10的多数为有益菌,并且成功分离得到16株芽孢杆菌属细菌,可为西山柴胡的生态种植提供理论基础。
基金Project supported by the National High Technology Development Program National Climbing Project
文摘A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfec-tion with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 amplicon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-liter rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.
文摘Background:Cryptococcal meningitis is a severe infectious disease associated with high morbidity and mortality.Rapidity and accuracy of diagnosis contribute to better prognosis,but readily available tools,such as microscopy,culture,and antigens do not perform well all the time.Our study attempted to diagnose and genotype cryptococcus in the cerebrospinal fluid(CSF)samples from patients with cryptococcal meningitis using the approach of metataxonomics of Internal Transcribed Spacer(ITS)amplicons.Methods:The CSF samples were collected from 11 clinically suspected cryptococcal meningitis patients and four non-infectious controls.Samples were recruited from the First Affiliated Hospital of Fujian Medical University Hospital,Fuzhou Fourth Hospital and the 476th Hospital of Chinese People's Liberation Army from December 2017 to December 2018.ITS1 ribosomal deoxyribonucleic acid(rDNA)genes of 15 whole samples were amplified by universal forward primer ITS1(CTTGGTCATTTAGAGGAAGTAA)and reverse primer ITS2(GCTGCGTTCTTCATCGATGC),sequenced by Illumina MiSeq Benchtop Sequencer.The results were confirmed by sanger sequencing of ITS1 region and partial CAP59 gene of microbial isolates from 11 meningitic samples.Pair-wise comparison between infectious group and control group was conducted through permutational multivariate analysis(PERMANOVA)in R software.Results:The 30,000 to 340,000 high-quality clean reads were obtained from each of the positively stained or cultured CSF samples and 8 to 60 reads from each control.The samples from 11 infected patients yielded detectable cryptococcal-specific ITS1 DNA with top abundance(from 95.90%to 99.97%),followed by many other fungal groups(each<1.41%).ITS genotype was defined in 11 CSF samples,corresponding to ITS type 1,and confirmed by Sanger sequencing.A statistically significant difference(r2=0.65869,P=0.0014)between infectious group and control group was observed.Conclusions:The metataxonomics of ITS amplicons facilitates the diagnosis and genotype of cryptococcus in CSF samples,which may provide a better diagnostic approach of cryptococcal infection.
基金This work was.financially supported by the Na-tional Natural Science Foundation of China(Grant No.31601698)the Natural Science Foundation of Zhejiang Province(Grant No.LR 19C140001)+1 种基金the Young Elite Scientist Sponsorship Program by CAST(Grant No.2016QNRC001)the Key Research and Development Program of Zhejiang Province(Grant No.2019C02015).
文摘The microbiome associated with brown planthopper(BPH)plays an important role in mediating host health and fitness.Characterization of the microbial community and its structure is prerequisite for understanding the intricate symbiotic relationships between microbes and host insect.Here,we investigated the bacterial and fungal communities of BPH at different developmental stages using high-throughput amplicon sequencing.Our results revealed that both the bacterial and fungal communities were diverse and dynamic during BPH development.The bacterial communities were generally richer than fungi in each developmental stage.At 97%similarly,19 phyla and 278 genera of bacteria were an-notated,while five fungal phyla comprising 80 genera were assigned.The highest species richness for the bacterial communities was detected in the nymphal stage.The taxonomic diversity of the fungal communities in female adults was generally at a relatively higher level when compared to other developmental stages.The most dominant phylum of bacteria and fungi at each developmental stage all belonged to Proteobacteria and Ascomycota,re-spectively.A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages,while the dominant fun-gal genus Wallemia was more abundant in the nymph and adult stages than in the egg stage.Additionally,the microbial composition differed between male and female adults,suggesting that the microbial communties In BPH were gender-dependent.Uverall,our study enriches our knowledge on the microbial communities associated with BPH and will provide clues to develop potential biocontrol techniques against this rice pest.
基金supported by the National Natural Science Foundation of China(31961123002,31872228)the Fundamental Research Funds for the Central Universities of China(202041001)+1 种基金the Young Taishan Scholars Program of Shandong Province(tsqn201812024)the National Science Foundation(DEB-1927159).
文摘High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.
