The 454 sequencing method was used to detect bacterial diversity and community structure in the East China Sea. Overall, 149 067 optimized reads with an average length of 454 nucleotides were obtained from 17 seawater...The 454 sequencing method was used to detect bacterial diversity and community structure in the East China Sea. Overall, 149 067 optimized reads with an average length of 454 nucleotides were obtained from 17 seawater samples and fi ve sediment samples sourced in May 2011. A total of 22 phyla, 34 classes, 74 orders, 146 families, and 333 genera were identifi ed in this study. Some of them were detected for the fi rst time from the East China Sea. The estimated richness and diversity indices were both higher in the sediment samples compared with in the seawater samples. All the samples were divided by their diversity indices into four regions. Similarity analysis showed that the seawater samples could be classifi ed into six groups. The groups differed from each other and had unique community structure characteristics. It was found that different water masses in the sampling areas may have had some infl uence on the bacterial community structure. A canonical correspondence analysis revealed that seawater samples in different areas and at different depths were affected by different environmental parameters. This study will lay the foundation for future research on microbiology in the East China Sea.展开更多
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
New terrestrial habitats have emerged and a primary succession has developed in the retreat area （29°34＇N, 102°oo＇E, 2951-2886 m） after the retreat of the Hailuogou glacier. To investigate soil microbial...New terrestrial habitats have emerged and a primary succession has developed in the retreat area （29°34＇N, 102°oo＇E, 2951-2886 m） after the retreat of the Hailuogou glacier. To investigate soil microbial changes along the primary successional chronosequence, mixed soil samples were collected at six sites at different ages （2 young sites, 2 mid-aged sites, and 2 old sites）. The RNA was extracted and amplified. Bacterial 16S rRNA and fungal 18S rRNA were analyzed using high-throughput 454 pyrosequencing analysis. Overall, pyrosequeneing showed that Proteobacteria, Acidobacteria, Baeteroidetes and Actinobacteria were the main bacterial phyla, and the fungal communities were strongly dominated by the phyla Ascomyeota and Basidiomyeota in the retreat area. The Shannon diversity index （Hshannon） of bacteria was 6.5 - 7.9, and that of fungi was 2.2 - 4.1 in these sites. For the bacterial communities, diversity and evenness values were highest on the mid-age sites and were relatively low on the young trend was observed for the and old sites. A similar fungal communities. In contrast, soil properties showed significant linear distributional trends （increase or decrease） with the age of the site. Combining the linear change patterns of soil properties, the highest values of bacterial and fungal evenness and diversity in the mid-aged sites indicated that there was less environmental stress and more niches for microbial communities in the middle successional stage compare with other stages. In addition, our analysis showed that microbial communities were the main drivers that build a soil organic matter pool to expedite pedogenesis for ecosystem succession. This primary succession in the Hailuogou glacier retreat area is developing rapidly compared with that in other glacier retreats.展开更多
The sea cucumberApostichopus japonicus is an important economic species in China. Its dorsal body wall color is commonly tawny, whereas its ventral surface is fawn. Albino sea cucumbers are rarely observed. In order t...The sea cucumberApostichopus japonicus is an important economic species in China. Its dorsal body wall color is commonly tawny, whereas its ventral surface is fawn. Albino sea cucumbers are rarely observed. In order to profile gene expression and screen albinism-related genes, we compared the transcriptome of albino samples with a control by 454 cDNA sequencing. We found that 6 539 identified genes on the basis of sequence similarity to known genes were expressed in the albino A. japonicus. The gene ontology analysis indicated that the transcription of genes associated with the terms of biological regulation and pigmenta-tion was non-abundant in the albino library compared to the control. Based on an analysis using the Kyoto Encyclopedia of Genes and Genomics （KEGG） database, we identified 14 important genes that were in-volved in major intercellular signaling pathways related to melanin synthesis, such as tyrosine metabolism, the mitogen-activated protein kinase （MAPK） pathway, and melanogenesis. The expressions of fibroblast growth factor receptor 4 （FGFR4）, protein kinase C （PKC）, protein kinase A （PKA）, and Ras genes were sig-nificantly down-regulated in the albino transcriptome compared with the control, while the expressions of homogentisate 1, 2-dioxygenase gene （HGO）, cAMP-responsive element binding protein （CREB）, transcrip-tion factor AP-1（c-jun）, and calmodulin （CaM） were significantly up-regulated （Fisher＇s exact test,p 〈 0.05）. These differentially expressed genes could be candidate genes for revealing the mechanism of albinism and investigating regulation of melanin synthesis inA. japonicus.展开更多
Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield. Gene enolase2 （EN02） is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant （enoZ） ...Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield. Gene enolase2 （EN02） is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant （enoZ） plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during NaCl treatment. Quantitative real-time RT-PCR （RT-qPCR） was performed to investigate the expression profile of EN02 in response to NaCl stress in Arabidopsis. The transcript level of EN02 was rapidly elevated in 300 mmol L-1 NaCl treatment. ENO2 also responded to 300 mmol L 1 NaCl treatment at the protein level. To illuminate the mechanism underlying EN02 resistance to salt at the transcriptional level, we studied the wild-type and enoZ Arabidopsis lines that were treated with 300 mmol L 1 NaCl 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 w-r-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, EN02 contributes to increased resistance to abiotic stress. In particular, EN02 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 EN02, 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 EN02 may regulate the expression of PGK, PGK1, TPI, and PK. Taken together, the results from this study reflects that EN02 gene has an important role in the response to the high salt stress.展开更多
Whole genome sequencing of buffalo is yet to be completed, and in the near future it may not be possible to identify an exome （coding region of genome） through bioinformatics for designing probes to capture it. In t...Whole genome sequencing of buffalo is yet to be completed, and in the near future it may not be possible to identify an exome （coding region of genome） through bioinformatics for designing probes to capture it. In the present study, we employed in solution hybridization to sequence tissue specific temporal exomes （TST exome） in buffalo. We utilized cDNA prepared from buffalo muscle tissue as a probe to capture TST exomes from the buffalo genome. This resulted in a prominent reduction of repeat sequences （up to 40%） and an enrichment of coding sequences （up to 60%）. Enriched targets were sequenced on a 454 pyro-sequencing platform, generating 101,244 reads contain- ing 24,127,779 high quality bases. The data revealed 40,100 variations, of which 403 were indels and 39,218 SNPs containing 195 nonsyn- onymous candidate SNPs in protein-coding regions. The study has indicated that 80% of the total genes identified from capture data were expressed in muscle tissue. The present study is the first of its kind to sequence TST exomes captured by use of cDNA molecules for SNPs found in the coding region without any prior sequence information of targeted molecules.展开更多
A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene ampl...A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene amplicons ('metabarcoding') have made it possible to survey biodiversity in a robust and efficient way. However, one obstacle to the widespread adoption of this technique is the need to choose amongst many candidates for bioinformatic processing of the raw sequencing data. We compare three candidate pipelines for the processing of 18S small subunit rDNA metabarcode data from solid substrates: (i) USEARCH/CROP, (ii) Denoiser/UCLUST, and (iii) OCTUPUS. The three pipelines produced reassuringly similar and highly correlated assessments of community composition that are dominated by taxa known to characterize the sampled environments. However, OCTUPUS appears to inflate phylogenetic diversity, because of higher sequence noise. We therefore recommend either the USEARCH/CROP or Denoiser/UCLUST pipelines, both of which can be run within the QIIME (Quantitative Insights Into Microbial Ecology) environment.展开更多
基金Supported by the National Basic Research Program of China(973 Program)(No.2011CB409804)the National Natural Science Foundation of China(No.41121064)
文摘The 454 sequencing method was used to detect bacterial diversity and community structure in the East China Sea. Overall, 149 067 optimized reads with an average length of 454 nucleotides were obtained from 17 seawater samples and fi ve sediment samples sourced in May 2011. A total of 22 phyla, 34 classes, 74 orders, 146 families, and 333 genera were identifi ed in this study. Some of them were detected for the fi rst time from the East China Sea. The estimated richness and diversity indices were both higher in the sediment samples compared with in the seawater samples. All the samples were divided by their diversity indices into four regions. Similarity analysis showed that the seawater samples could be classifi ed into six groups. The groups differed from each other and had unique community structure characteristics. It was found that different water masses in the sampling areas may have had some infl uence on the bacterial community structure. A canonical correspondence analysis revealed that seawater samples in different areas and at different depths were affected by different environmental parameters. This study will lay the foundation for future research on microbiology in the East China Sea.
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(Grant Nos.41501281 and 41272200)the Chinese Academy of Sciences(CAS "Light of West China" Program)
文摘New terrestrial habitats have emerged and a primary succession has developed in the retreat area （29°34＇N, 102°oo＇E, 2951-2886 m） after the retreat of the Hailuogou glacier. To investigate soil microbial changes along the primary successional chronosequence, mixed soil samples were collected at six sites at different ages （2 young sites, 2 mid-aged sites, and 2 old sites）. The RNA was extracted and amplified. Bacterial 16S rRNA and fungal 18S rRNA were analyzed using high-throughput 454 pyrosequencing analysis. Overall, pyrosequeneing showed that Proteobacteria, Acidobacteria, Baeteroidetes and Actinobacteria were the main bacterial phyla, and the fungal communities were strongly dominated by the phyla Ascomyeota and Basidiomyeota in the retreat area. The Shannon diversity index （Hshannon） of bacteria was 6.5 - 7.9, and that of fungi was 2.2 - 4.1 in these sites. For the bacterial communities, diversity and evenness values were highest on the mid-age sites and were relatively low on the young trend was observed for the and old sites. A similar fungal communities. In contrast, soil properties showed significant linear distributional trends （increase or decrease） with the age of the site. Combining the linear change patterns of soil properties, the highest values of bacterial and fungal evenness and diversity in the mid-aged sites indicated that there was less environmental stress and more niches for microbial communities in the middle successional stage compare with other stages. In addition, our analysis showed that microbial communities were the main drivers that build a soil organic matter pool to expedite pedogenesis for ecosystem succession. This primary succession in the Hailuogou glacier retreat area is developing rapidly compared with that in other glacier retreats.
基金The National Natural Science Foundation of China under contract No.40976089the State Science&Technology Support Program in the 12th Five Year Plan of China under contract No.2011BAD13B05the National High-tech R&D Program of China(863 Program) under contract No.2012AA10A412
文摘The sea cucumberApostichopus japonicus is an important economic species in China. Its dorsal body wall color is commonly tawny, whereas its ventral surface is fawn. Albino sea cucumbers are rarely observed. In order to profile gene expression and screen albinism-related genes, we compared the transcriptome of albino samples with a control by 454 cDNA sequencing. We found that 6 539 identified genes on the basis of sequence similarity to known genes were expressed in the albino A. japonicus. The gene ontology analysis indicated that the transcription of genes associated with the terms of biological regulation and pigmenta-tion was non-abundant in the albino library compared to the control. Based on an analysis using the Kyoto Encyclopedia of Genes and Genomics （KEGG） database, we identified 14 important genes that were in-volved in major intercellular signaling pathways related to melanin synthesis, such as tyrosine metabolism, the mitogen-activated protein kinase （MAPK） pathway, and melanogenesis. The expressions of fibroblast growth factor receptor 4 （FGFR4）, protein kinase C （PKC）, protein kinase A （PKA）, and Ras genes were sig-nificantly down-regulated in the albino transcriptome compared with the control, while the expressions of homogentisate 1, 2-dioxygenase gene （HGO）, cAMP-responsive element binding protein （CREB）, transcrip-tion factor AP-1（c-jun）, and calmodulin （CaM） were significantly up-regulated （Fisher＇s exact test,p 〈 0.05）. These differentially expressed genes could be candidate genes for revealing the mechanism of albinism and investigating regulation of melanin synthesis inA. japonicus.
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 （EN02） is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant （enoZ） plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during NaCl treatment. Quantitative real-time RT-PCR （RT-qPCR） was performed to investigate the expression profile of EN02 in response to NaCl stress in Arabidopsis. The transcript level of EN02 was rapidly elevated in 300 mmol L-1 NaCl treatment. ENO2 also responded to 300 mmol L 1 NaCl treatment at the protein level. To illuminate the mechanism underlying EN02 resistance to salt at the transcriptional level, we studied the wild-type and enoZ Arabidopsis lines that were treated with 300 mmol L 1 NaCl 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 w-r-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, EN02 contributes to increased resistance to abiotic stress. In particular, EN02 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 EN02, 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 EN02 may regulate the expression of PGK, PGK1, TPI, and PK. Taken together, the results from this study reflects that EN02 gene has an important role in the response to the high salt stress.
文摘Whole genome sequencing of buffalo is yet to be completed, and in the near future it may not be possible to identify an exome （coding region of genome） through bioinformatics for designing probes to capture it. In the present study, we employed in solution hybridization to sequence tissue specific temporal exomes （TST exome） in buffalo. We utilized cDNA prepared from buffalo muscle tissue as a probe to capture TST exomes from the buffalo genome. This resulted in a prominent reduction of repeat sequences （up to 40%） and an enrichment of coding sequences （up to 60%）. Enriched targets were sequenced on a 454 pyro-sequencing platform, generating 101,244 reads contain- ing 24,127,779 high quality bases. The data revealed 40,100 variations, of which 403 were indels and 39,218 SNPs containing 195 nonsyn- onymous candidate SNPs in protein-coding regions. The study has indicated that 80% of the total genes identified from capture data were expressed in muscle tissue. The present study is the first of its kind to sequence TST exomes captured by use of cDNA molecules for SNPs found in the coding region without any prior sequence information of targeted molecules.
基金supported by Yunnan Province (20080A001)Chinese Academy of Sciences (0902281081,KSCX2-YW-Z-1027)+2 种基金the National Natural Science Foundation of China (31170498)Ministry of Science and Technology of China (2012FY110800)Kunming Institute of Zoology,and the University of East Anglia
文摘A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene amplicons ('metabarcoding') have made it possible to survey biodiversity in a robust and efficient way. However, one obstacle to the widespread adoption of this technique is the need to choose amongst many candidates for bioinformatic processing of the raw sequencing data. We compare three candidate pipelines for the processing of 18S small subunit rDNA metabarcode data from solid substrates: (i) USEARCH/CROP, (ii) Denoiser/UCLUST, and (iii) OCTUPUS. The three pipelines produced reassuringly similar and highly correlated assessments of community composition that are dominated by taxa known to characterize the sampled environments. However, OCTUPUS appears to inflate phylogenetic diversity, because of higher sequence noise. We therefore recommend either the USEARCH/CROP or Denoiser/UCLUST pipelines, both of which can be run within the QIIME (Quantitative Insights Into Microbial Ecology) environment.
