AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using ...AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.展开更多
Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield.Gene enolase2(ENO2)is important in resistance to abiotic stress in various organisms.ENO2 T-DNA insertion mutant(eno2~–)plants ...Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield.Gene enolase2(ENO2)is important in resistance to abiotic stress in various organisms.ENO2 T-DNA insertion mutant(eno2~–)plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during Na Cl treatment.Quantitative real-time RT-PCR(RT-q PCR)was performed to investigate the expression profile of ENO2 in response to Na Cl stress in Arabidopsis.The transcript level of ENO2 was rapidly elevated in 300 mmol L^(–1) Na Cl treatment.ENO2 also responded to 300 mmol L^(–1) Na Cl treatment at the protein level.To illuminate the mechanism underlying ENO2 resistance to salt at the transcriptional level,we studied the wild-type and eno2~–Arabidopsis lines that were treated with 300 mmol L^(–1) Na Cl for 18 h using 454 GS FLX,which resulted in an expressed sequence tag(EST)dataset.A total of 961 up-regulated and 746 down-regulated differentially expressed genes(DEGs)were identified in the pairwise comparison WT-18 h:eno2~–-18 h.The DEGs were identified and functionally annotated using the databases of Gene Ontology(GO)and the Kyoto encyclopedia of genes and genomes(KEGG).The identified unigenes were subjected to GO analysis to determine biological,molecular,and cellular functions.The biological process was enriched in a total of 20 GO terms,the cellular component was enriched in 13 GO terms,and the molecular function was enriched in 11 GO terms.Using KEGG mapping,DEGs with pathway annotations contributed to 115 pathways.The top 3 pathways based on a statistical analysis were biosynthesis of the secondary metabolites(KO01110),plant-pathogen interactions(KO04626),and plant hormone signal transduction(KO04075).Based on these results,ENO2 contributes to increased resistance to abiotic stress.In particular,ENO2 is involved in some of the metabolic stress response pathways in Arabidopsis.Our work also demonstrates that this EST dataset will be a powerful resource for further studies of ENO2,such as functional analyses,investigations of biological roles,and molecular breeding.Additionally,3-phosphoglycerate kinase(PGK),3-phosphoglycerate kinase 1(PGK1),triosephosphate isomerase(TPI),and pyruvate kinase(PK)in glycolysis interactions with ENO2 were verified using the yeast two-hybrid experiment,and ENO2 may regulate the expression of PGK,PGK1,TPI,and PK.Taken together,the results from this study reflects that ENO2 gene has an important role in the response to the high salt stress.展开更多
基金Supported by(in part)Grants UH2CA140233 from the Human Microbiome Project of the NIH Roadmap Initiative and National Cancer InstituteR01AI063477 from the National Institute of Allergy and Infectious Diseases+1 种基金DE-11443 from the National Institute of Dental and Craniofacial ResearchU19DE018385 from the National Institute of Dental & Craniofacial Research
文摘AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.
基金funded by the National Natural Science Foundation of China (31470399 and 31270365)
文摘Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield.Gene enolase2(ENO2)is important in resistance to abiotic stress in various organisms.ENO2 T-DNA insertion mutant(eno2~–)plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during Na Cl treatment.Quantitative real-time RT-PCR(RT-q PCR)was performed to investigate the expression profile of ENO2 in response to Na Cl stress in Arabidopsis.The transcript level of ENO2 was rapidly elevated in 300 mmol L^(–1) Na Cl treatment.ENO2 also responded to 300 mmol L^(–1) Na Cl treatment at the protein level.To illuminate the mechanism underlying ENO2 resistance to salt at the transcriptional level,we studied the wild-type and eno2~–Arabidopsis lines that were treated with 300 mmol L^(–1) Na Cl for 18 h using 454 GS FLX,which resulted in an expressed sequence tag(EST)dataset.A total of 961 up-regulated and 746 down-regulated differentially expressed genes(DEGs)were identified in the pairwise comparison WT-18 h:eno2~–-18 h.The DEGs were identified and functionally annotated using the databases of Gene Ontology(GO)and the Kyoto encyclopedia of genes and genomes(KEGG).The identified unigenes were subjected to GO analysis to determine biological,molecular,and cellular functions.The biological process was enriched in a total of 20 GO terms,the cellular component was enriched in 13 GO terms,and the molecular function was enriched in 11 GO terms.Using KEGG mapping,DEGs with pathway annotations contributed to 115 pathways.The top 3 pathways based on a statistical analysis were biosynthesis of the secondary metabolites(KO01110),plant-pathogen interactions(KO04626),and plant hormone signal transduction(KO04075).Based on these results,ENO2 contributes to increased resistance to abiotic stress.In particular,ENO2 is involved in some of the metabolic stress response pathways in Arabidopsis.Our work also demonstrates that this EST dataset will be a powerful resource for further studies of ENO2,such as functional analyses,investigations of biological roles,and molecular breeding.Additionally,3-phosphoglycerate kinase(PGK),3-phosphoglycerate kinase 1(PGK1),triosephosphate isomerase(TPI),and pyruvate kinase(PK)in glycolysis interactions with ENO2 were verified using the yeast two-hybrid experiment,and ENO2 may regulate the expression of PGK,PGK1,TPI,and PK.Taken together,the results from this study reflects that ENO2 gene has an important role in the response to the high salt stress.
