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Illumina Inc deep sequencing
Deep Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Amplification:

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Therefore, mutants at roughly 0.1% represent intrinsic errors of sequencing procedures and mutations introduced by PCR amplification. .. To study this phenomenon, we analyzed a set of eight poliovirus samples by both pyrosequencing and Illumina methods.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: The background level of mutations produced by PCR amplification and base-calling errors inherent to any sequencing technology puts a lower limit on the number of mutations that could be detected by this method. .. Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants.

Article Title: DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis
Article Snippet: Samples were amplified by the bisulphite–polymerase chain reaction (PCR) (HotStar Taq Polymerase, Qiagen) and sequenced using the Pyrosequencing PSQ96 HS System (Qiagen) with an assay developed by Epigendx. .. Correlation between DNA methylation levels at cg01476222 CpG sites estimated by pyrosequencing and the Illumina 450K BeadChip array was calculated using Spearman’s rank correlation coefficient.

High Throughput Screening Assay:

Article Title: Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function
Article Snippet: To prepare samples for ChIPSeq, the immunoprecipitated DNA was isolated and DNA fragments (200 ± 25 bp) were gel purified by Ambry Genetics (Aliso Viejo, CA). .. Approximately 10 ng of TRP120 and control ChIP DNA was sequenced by high-throughput DNA sequencing according to the library preparation protocol from Illumina using a Solexa/Illumina genome analyzer (Ambry Genetics). .. All sequence reads produced by the Illumina genome analyzer were analyzed by base calling and sequence quality filtering scripts using the Illumina Pipeline software (version 1.4.0; Illumina, Hayward, CA).

Article Title: microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition
Article Snippet: Functionally, miR-181a inhibition led to a decrease in cellular survival ( ) and rendered the cells less migratory ( ). .. To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis. .. Using a threshold of a minimum fold change > 5 or fold change < 5 (P < 0.05, Fisher exact test,) we identified a total of 739 differentially expressed genes: 705 downregulated and 34 upregulated ( ).

Article Title: Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation
Article Snippet: Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max ) near-isogenic line (cgy-2- NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. .. To identify α-null-related transcriptional changes, the gene expressions of cgy-2- NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF). .. Seeds at 18 DAF served as the control.

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: This library was selected against the target SA and also subjected to a “no-protein” control selection, similar to that for , to control for biases from the selection procedures (irradiation, gel purification, PCR, sequencing, etc.). .. The selection results were decoded by high throughput DNA sequencing (Illumina®). .. Again, due to the high affinity of the LD-5 / AD-5 duplex, the sequence that encodes LD-5 was distinctly enriched (19.2-fold).

Real-time Polymerase Chain Reaction:

Article Title: Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials
Article Snippet: These results are consistent with our visual observations of DNA quality as well as with the decrease in sequence quality obtained and illustrate that irradiated material can be detected via PCR, yet the sensitivity is sacrificed ( , ). .. In contrast to the requirements for longer DNA fragments during 454 pyrosequencing and Illumina library preparation, qPCR detection schemes typically amplify fragments of less than 140 bp (131 bp for B. atrophaeus variant globigii and 67 bp for Y. pestis in this study). .. The probability of occurrence of radiation damage in any given fragment increases with the strand length; therefore, it is not surprising that methods that are dependent on smaller fragments would be less affected by irradiation than those that require longer strands.

Microarray:

Article Title: Differentially methylated regions in T cells identify kidney transplant patients at risk for de novo skin cancer
Article Snippet: Data processing and statistical analysis of all the microarray data was done in RStudio version 1.0.136 (Rstudio Inc., Boston, MA, US) with R version 3.2.5 [ ]. .. Cohen’s D was calculated on the residuals of the linear mixed effect model by the formula D = (meancSCC − meannon-cSCC )/sdpooled in R. Analysis of the differences between methylation in pre-transplantation and post-transplantation samples was done using a paired Wilcoxon ranked sum test using R. Correlation between the DNA methylation levels quantified by pyrosequencing and the beta-values of the Illumina 450k arrays was calculated using Spearman’s rank correlation coefficient using SPSS.

Sampling:

Article Title: Host and Environmental Factors Affecting the Intestinal Microbiota in Chickens
Article Snippet: In contrast, in another recent study a relative abundance of 22% of Bacteroidetes was reported in the ileum of 18-day-old Ross broilers ( ). .. The presence of Bacteroidetes in the latter study and absence in the other studies may be caused by inevitable differences in diet or other experimental conditions, the younger age at sampling, differences in sequencing technology; as pyrosequencing vs. Illumina MiSeq, or the differences in the primers used. .. We compiled the data of studies for which 16S rRNA gene amplicon sequencing data of cecal samples was available for two broiler breeds ( Figures , ).

Gel Purification:

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: This library was selected against the target SA and also subjected to a “no-protein” control selection, similar to that for , to control for biases from the selection procedures (irradiation, gel purification, PCR, sequencing, etc.). .. The selection results were decoded by high throughput DNA sequencing (Illumina®).

MANN-WHITNEY:

Article Title: Differentially methylated regions in T cells identify kidney transplant patients at risk for de novo skin cancer
Article Snippet: The Mann-Whitney U test was used for the continuous variables and χ 2 test for the categorical variables. .. Cohen’s D was calculated on the residuals of the linear mixed effect model by the formula D = (meancSCC − meannon-cSCC )/sdpooled in R. Analysis of the differences between methylation in pre-transplantation and post-transplantation samples was done using a paired Wilcoxon ranked sum test using R. Correlation between the DNA methylation levels quantified by pyrosequencing and the beta-values of the Illumina 450k arrays was calculated using Spearman’s rank correlation coefficient using SPSS.

Immunoprecipitation:

Article Title: Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function
Article Snippet: To prepare samples for ChIPSeq, the immunoprecipitated DNA was isolated and DNA fragments (200 ± 25 bp) were gel purified by Ambry Genetics (Aliso Viejo, CA). .. Approximately 10 ng of TRP120 and control ChIP DNA was sequenced by high-throughput DNA sequencing according to the library preparation protocol from Illumina using a Solexa/Illumina genome analyzer (Ambry Genetics).

Infection:

Article Title: Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function
Article Snippet: Chromatin immunoprecipitation (ChIP) was performed on highly infected (95%) THP-1 cells 4 days after infection using a ChIP-IT Express kit (Active Motif, Carlsbad, CA) with anti- E. chaffeensis TRP120 antibody or preimmune serum from the same rabbit according to the manufacturer's instructions. .. Approximately 10 ng of TRP120 and control ChIP DNA was sequenced by high-throughput DNA sequencing according to the library preparation protocol from Illumina using a Solexa/Illumina genome analyzer (Ambry Genetics).

Generated:

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Two versions of MPS were used in this study: pyrosequencing (454 Life Sciences, a Roche company) and Illumina methods. .. Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants. .. Although the accuracy of base calls was roughly similar, pyrosequencing erroneously identified a significant number of insertions and deletions, especially in regions where the same base is repeated several times.

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: Metagenomic datasets were simulated for Sanger sequencing, pyrosequencing, and Illumina sequencing. .. For each sequencing technology, three metagenomes were simulated to mimic different community complexities (10, 100 and 400 genomes). ( , , , ).

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: As shown in , in all cases, the “TTT” codon encoding the desthiobiotin in LD-4 has been enriched markedly by SA due to the high affinity of the LD-4 / AD-4 duplex (left panels), whereas negative selections (no protein) only generated scrambled sequences at the encoding site ( , right panels). .. The selection results were decoded by high throughput DNA sequencing (Illumina®).

DNA Sequencing:

Article Title: Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function
Article Snippet: To prepare samples for ChIPSeq, the immunoprecipitated DNA was isolated and DNA fragments (200 ± 25 bp) were gel purified by Ambry Genetics (Aliso Viejo, CA). .. Approximately 10 ng of TRP120 and control ChIP DNA was sequenced by high-throughput DNA sequencing according to the library preparation protocol from Illumina using a Solexa/Illumina genome analyzer (Ambry Genetics). .. All sequence reads produced by the Illumina genome analyzer were analyzed by base calling and sequence quality filtering scripts using the Illumina Pipeline software (version 1.4.0; Illumina, Hayward, CA).

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: This library was selected against the target SA and also subjected to a “no-protein” control selection, similar to that for , to control for biases from the selection procedures (irradiation, gel purification, PCR, sequencing, etc.). .. The selection results were decoded by high throughput DNA sequencing (Illumina®). .. Again, due to the high affinity of the LD-5 / AD-5 duplex, the sequence that encodes LD-5 was distinctly enriched (19.2-fold).

Polymerase Chain Reaction:

Article Title: Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials
Article Snippet: These results are consistent with our visual observations of DNA quality as well as with the decrease in sequence quality obtained and illustrate that irradiated material can be detected via PCR, yet the sensitivity is sacrificed ( , ). .. In contrast to the requirements for longer DNA fragments during 454 pyrosequencing and Illumina library preparation, qPCR detection schemes typically amplify fragments of less than 140 bp (131 bp for B. atrophaeus variant globigii and 67 bp for Y. pestis in this study).

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Therefore, mutants at roughly 0.1% represent intrinsic errors of sequencing procedures and mutations introduced by PCR amplification. .. To study this phenomenon, we analyzed a set of eight poliovirus samples by both pyrosequencing and Illumina methods.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: The background level of mutations produced by PCR amplification and base-calling errors inherent to any sequencing technology puts a lower limit on the number of mutations that could be detected by this method. .. Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants.

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: This library was selected against the target SA and also subjected to a “no-protein” control selection, similar to that for , to control for biases from the selection procedures (irradiation, gel purification, PCR, sequencing, etc.). .. The selection results were decoded by high throughput DNA sequencing (Illumina®).

Article Title: DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis
Article Snippet: Samples were amplified by the bisulphite–polymerase chain reaction (PCR) (HotStar Taq Polymerase, Qiagen) and sequenced using the Pyrosequencing PSQ96 HS System (Qiagen) with an assay developed by Epigendx. .. Correlation between DNA methylation levels at cg01476222 CpG sites estimated by pyrosequencing and the Illumina 450K BeadChip array was calculated using Spearman’s rank correlation coefficient.

RNA Sequencing Assay:

Article Title: microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition
Article Snippet: Functionally, miR-181a inhibition led to a decrease in cellular survival ( ) and rendered the cells less migratory ( ). .. To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis. .. Using a threshold of a minimum fold change > 5 or fold change < 5 (P < 0.05, Fisher exact test,) we identified a total of 739 differentially expressed genes: 705 downregulated and 34 upregulated ( ).

Article Title: Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation
Article Snippet: Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max ) near-isogenic line (cgy-2- NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. .. To identify α-null-related transcriptional changes, the gene expressions of cgy-2- NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF). .. Seeds at 18 DAF served as the control.

Methylation:

Article Title: DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis
Article Snippet: The percentage of methylation was calculated for each CpG site as methylated cytosine divided by the sum of methylated and unmethylated cytosines. .. Correlation between DNA methylation levels at cg01476222 CpG sites estimated by pyrosequencing and the Illumina 450K BeadChip array was calculated using Spearman’s rank correlation coefficient.

Article Title: Differentially methylated regions in T cells identify kidney transplant patients at risk for de novo skin cancer
Article Snippet: Data processing and statistical analysis of all the microarray data was done in RStudio version 1.0.136 (Rstudio Inc., Boston, MA, US) with R version 3.2.5 [ ]. .. Cohen’s D was calculated on the residuals of the linear mixed effect model by the formula D = (meancSCC − meannon-cSCC )/sdpooled in R. Analysis of the differences between methylation in pre-transplantation and post-transplantation samples was done using a paired Wilcoxon ranked sum test using R. Correlation between the DNA methylation levels quantified by pyrosequencing and the beta-values of the Illumina 450k arrays was calculated using Spearman’s rank correlation coefficient using SPSS. .. All statistical tests were two-tailed, and a p < 0.05 was considered statistically significant.

Mutagenesis:

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina. .. Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina.

Isolation:

Article Title: Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials
Article Snippet: The cycle threshold ( CT ) values obtained from irradiated and control material were compared, albeit irradiation had detectable effects on the integrity of the DNA isolated from vegetative cells. .. In contrast to the requirements for longer DNA fragments during 454 pyrosequencing and Illumina library preparation, qPCR detection schemes typically amplify fragments of less than 140 bp (131 bp for B. atrophaeus variant globigii and 67 bp for Y. pestis in this study).

Article Title: Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function
Article Snippet: To prepare samples for ChIPSeq, the immunoprecipitated DNA was isolated and DNA fragments (200 ± 25 bp) were gel purified by Ambry Genetics (Aliso Viejo, CA). .. Approximately 10 ng of TRP120 and control ChIP DNA was sequenced by high-throughput DNA sequencing according to the library preparation protocol from Illumina using a Solexa/Illumina genome analyzer (Ambry Genetics).

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: The selection results were decoded by high throughput DNA sequencing (Illumina®). .. The selection results were decoded by high throughput DNA sequencing (Illumina®).

Purification:

Article Title: Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function
Article Snippet: To prepare samples for ChIPSeq, the immunoprecipitated DNA was isolated and DNA fragments (200 ± 25 bp) were gel purified by Ambry Genetics (Aliso Viejo, CA). .. Approximately 10 ng of TRP120 and control ChIP DNA was sequenced by high-throughput DNA sequencing according to the library preparation protocol from Illumina using a Solexa/Illumina genome analyzer (Ambry Genetics).

Sequencing:

Article Title: Host and Environmental Factors Affecting the Intestinal Microbiota in Chickens
Article Snippet: In contrast, in another recent study a relative abundance of 22% of Bacteroidetes was reported in the ileum of 18-day-old Ross broilers ( ). .. The presence of Bacteroidetes in the latter study and absence in the other studies may be caused by inevitable differences in diet or other experimental conditions, the younger age at sampling, differences in sequencing technology; as pyrosequencing vs. Illumina MiSeq, or the differences in the primers used. .. We compiled the data of studies for which 16S rRNA gene amplicon sequencing data of cecal samples was available for two broiler breeds ( Figures , ).

Article Title: Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials
Article Snippet: These results are consistent with our visual observations of DNA quality as well as with the decrease in sequence quality obtained and illustrate that irradiated material can be detected via PCR, yet the sensitivity is sacrificed ( , ). .. In contrast to the requirements for longer DNA fragments during 454 pyrosequencing and Illumina library preparation, qPCR detection schemes typically amplify fragments of less than 140 bp (131 bp for B. atrophaeus variant globigii and 67 bp for Y. pestis in this study).

Article Title: microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition
Article Snippet: To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis. .. To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis.

Article Title: Metatranscriptome of an Anaerobic Benzene-Degrading, Nitrate-Reducing Enrichment Culture Reveals Involvement of Carboxylation in Benzene Ring Activation
Article Snippet: Functional analysis of the sequence data as detailed below revealed the value in sequencing RNA even without a reference metagenome, because many contigs with functional genes of interest were successfully assembled and could be annotated, providing insights into the dynamics of the culture on the two substrates tested, as described below. .. The total numbers of RNA and non-rRNA sequence reads obtained from pyrosequencing and Illumina protocols are summarized in . .. Illumina sequencing of the RNA samples (amended with benzene first and then with benzoate) generated 100 times more reads than the RNA samples sequenced via pyrosequencing (amended with benzoate first and then with benzene).

Article Title: Metatranscriptome of an Anaerobic Benzene-Degrading, Nitrate-Reducing Enrichment Culture Reveals Involvement of Carboxylation in Benzene Ring Activation
Article Snippet: Supplemental material: .. We thank the Center for Applied Genomics, Toronto, Canada for assistance with both pyrosequencing and Illumina sequencing. .. Funding was provided by the Government of Canada through NSERC, Genome Canada, and the Ontario Genomics Institute (grant 2009-OGI-ABC-1405 ) and by the Government of Ontario through the ORF-GL2 program.

Article Title: Random X inactivation in the mule and horse placenta
Article Snippet: IIlumina RNA-seq data in this study have been submitted to the NCBI Gene Expression Omnibus (GEO) ( ) under accession no. . .. We thank Jenny Xiang, Ying (Diana) Shao, Amanda Manfredo, and Li (Grace) Chi for assistance with Illumina sequencing and pyrosequencing experiments. .. We also thank Jim Hardy, Scott Hoffay, and Emily Silvela for management of the Baker Institute equid herd.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Mutants in excess of this level may reflect the true sequence heterogeneity of viral quasispecies. .. To study this phenomenon, we analyzed a set of eight poliovirus samples by both pyrosequencing and Illumina methods.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: To study this phenomenon, we analyzed a set of eight poliovirus samples by both pyrosequencing and Illumina methods. .. Various characteristics of both datasets shown in demonstrate that the average sequence length was significantly higher for pyrosequencing (195 vs. 38), but the Illumina method produced roughly 60 times more sequence information. .. Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Various characteristics of both datasets shown in demonstrate that the average sequence length was significantly higher for pyrosequencing (195 vs. 38), but the Illumina method produced roughly 60 times more sequence information. .. Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Two versions of MPS were used in this study: pyrosequencing (454 Life Sciences, a Roche company) and Illumina methods. .. Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants. .. Although the accuracy of base calls was roughly similar, pyrosequencing erroneously identified a significant number of insertions and deletions, especially in regions where the same base is repeated several times.

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: The contig score can vary between 0 and 100, with 100 being the best value. .. The percentage of chimeric contigs was lowest in Sanger sequencing, while pyrosequencing and Illumina had a much higher percentage of chimeric contigs ( , ). .. However, the degree of chimericity (percentage of reads from “wrong” genomes ) was on average higher for Sanger sequencing than the other sequencing technologies, with the effect being more pronounced in the least complex community, and almost negligible in the most complex community.

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: The sequences and their assigned quality values are returned as fastq formatted files . .. Metagenomic datasets were simulated for Sanger sequencing, pyrosequencing, and Illumina sequencing. .. For each sequencing technology, three metagenomes were simulated to mimic different community complexities (10, 100 and 400 genomes). ( , , , ).

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: For the most complex community, where there was low coverage of each genome, assemblies from Illumina and pyrosequencing failed to represent the expected functional composition of the metagenomes, as there were very few complete genes annotated. .. For the most complex community, where there was low coverage of each genome, assemblies from Illumina and pyrosequencing failed to represent the expected functional composition of the metagenomes, as there were very few complete genes annotated.

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: For the more complex community (100 genomes), the Illumina sequences assembled a much greater length of contigs resulting in more complete genes. .. This is due to the greater sequencing depth achieved as the price per base is much less for Illumina sequencing than pyrosequencing or Sanger. .. For the most complex community none of the sequencing technologies assembled many reads into contigs.

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: This library was selected against the target SA and also subjected to a “no-protein” control selection, similar to that for , to control for biases from the selection procedures (irradiation, gel purification, PCR, sequencing, etc.). .. The selection results were decoded by high throughput DNA sequencing (Illumina®).

Article Title: Chromosome-wide profiling of X-chromosome inactivation and epigenetic states in fetal brain and placenta of the opossum, Monodelphis domestica
Article Snippet: Development of the M. domestica stocks was supported by a grant to J.L.V. from the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation. .. We thank Jenny Xiang, Ying (Diana) Shao, Wei Zhang, Amanda Manfredo, and Li (Grace) Chi for assistance with Illumina sequencing and pyrosequencing experiments. .. We also thank Madhu Jasti and Abigail Wolff for assistance with ChIP and genotyping experiments.

Polyacrylamide Gel Electrophoresis:

Article Title: Chromosome-wide profiling of X-chromosome inactivation and epigenetic states in fetal brain and placenta of the opossum, Monodelphis domestica
Article Snippet: We thank Jenny Xiang, Ying (Diana) Shao, Wei Zhang, Amanda Manfredo, and Li (Grace) Chi for assistance with Illumina sequencing and pyrosequencing experiments. .. We also thank Madhu Jasti and Abigail Wolff for assistance with ChIP and genotyping experiments.

Chromatin Immunoprecipitation:

Article Title: Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function
Article Snippet: To prepare samples for ChIPSeq, the immunoprecipitated DNA was isolated and DNA fragments (200 ± 25 bp) were gel purified by Ambry Genetics (Aliso Viejo, CA). .. Approximately 10 ng of TRP120 and control ChIP DNA was sequenced by high-throughput DNA sequencing according to the library preparation protocol from Illumina using a Solexa/Illumina genome analyzer (Ambry Genetics). .. All sequence reads produced by the Illumina genome analyzer were analyzed by base calling and sequence quality filtering scripts using the Illumina Pipeline software (version 1.4.0; Illumina, Hayward, CA).

Plasmid Preparation:

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: The rate of mutations in the PCR amplicon produced from the plasmid was higher (about 0.1%). .. Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants.

Software:

Article Title: microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition
Article Snippet: To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis. .. To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants. .. Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants.

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: In addition, most Illumina assembly software allows reads to be assigned to more than one contig. .. The percentage of chimeric contigs was lowest in Sanger sequencing, while pyrosequencing and Illumina had a much higher percentage of chimeric contigs ( , ).

Irradiation:

Article Title: Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials
Article Snippet: Paragraph title: Effects of irradiation on qPCR detection. ... In contrast to the requirements for longer DNA fragments during 454 pyrosequencing and Illumina library preparation, qPCR detection schemes typically amplify fragments of less than 140 bp (131 bp for B. atrophaeus variant globigii and 67 bp for Y. pestis in this study).

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: This library was selected against the target SA and also subjected to a “no-protein” control selection, similar to that for , to control for biases from the selection procedures (irradiation, gel purification, PCR, sequencing, etc.). .. The selection results were decoded by high throughput DNA sequencing (Illumina®).

Functional Assay:

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: This is also because the amount of sequence produced allowed for all of the sequencing technologies to provide enough coverage of each genome. .. For the most complex community, where there was low coverage of each genome, assemblies from Illumina and pyrosequencing failed to represent the expected functional composition of the metagenomes, as there were very few complete genes annotated. .. However, the Sanger reads approximated the expected functional composition reasonably well as the length of the reads allowed for accurate functional annotation.

Selection:

Article Title: Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation
Article Snippet: Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max ) near-isogenic line (cgy-2- NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. .. To identify α-null-related transcriptional changes, the gene expressions of cgy-2- NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF).

Article Title: Design, preparation, and selection of DNA-encoded dynamic libraries †Electronic supplementary information (ESI) available: Materials and general methods, experimental details, library selection and sequencing methods and fold enrichment calculations. See DOI: 10.1039/c5sc02467fClick here for additional data file.
Article Snippet: This library was selected against the target SA and also subjected to a “no-protein” control selection, similar to that for , to control for biases from the selection procedures (irradiation, gel purification, PCR, sequencing, etc.). .. The selection results were decoded by high throughput DNA sequencing (Illumina®). .. Again, due to the high affinity of the LD-5 / AD-5 duplex, the sequence that encodes LD-5 was distinctly enriched (19.2-fold).

Next-Generation Sequencing:

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: The percentage of chimeric contigs was lowest in Sanger sequencing, while pyrosequencing and Illumina had a much higher percentage of chimeric contigs ( , ). .. The percentage of chimeric contigs was lowest in Sanger sequencing, while pyrosequencing and Illumina had a much higher percentage of chimeric contigs ( , ).

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: For the most complex community, where there was low coverage of each genome, assemblies from Illumina and pyrosequencing failed to represent the expected functional composition of the metagenomes, as there were very few complete genes annotated. .. For the most complex community, where there was low coverage of each genome, assemblies from Illumina and pyrosequencing failed to represent the expected functional composition of the metagenomes, as there were very few complete genes annotated.

DNA Methylation Assay:

Article Title: DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis
Article Snippet: The percentage of methylation was calculated for each CpG site as methylated cytosine divided by the sum of methylated and unmethylated cytosines. .. Correlation between DNA methylation levels at cg01476222 CpG sites estimated by pyrosequencing and the Illumina 450K BeadChip array was calculated using Spearman’s rank correlation coefficient. .. RNA was extracted from the PBMCs of IBD patients ( n = 63) and controls ( n = 20) using an E.Z.N.A.® RNA Isolation Kit (VWR, Radnor, PA) and converted to cDNA (OmniScript, Qiagen) following the manufacturer’s instructions.

Article Title: Differentially methylated regions in T cells identify kidney transplant patients at risk for de novo skin cancer
Article Snippet: Data processing and statistical analysis of all the microarray data was done in RStudio version 1.0.136 (Rstudio Inc., Boston, MA, US) with R version 3.2.5 [ ]. .. Cohen’s D was calculated on the residuals of the linear mixed effect model by the formula D = (meancSCC − meannon-cSCC )/sdpooled in R. Analysis of the differences between methylation in pre-transplantation and post-transplantation samples was done using a paired Wilcoxon ranked sum test using R. Correlation between the DNA methylation levels quantified by pyrosequencing and the beta-values of the Illumina 450k arrays was calculated using Spearman’s rank correlation coefficient using SPSS. .. All statistical tests were two-tailed, and a p < 0.05 was considered statistically significant.

Produced:

Article Title: Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation
Article Snippet: Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max ) near-isogenic line (cgy-2- NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. .. To identify α-null-related transcriptional changes, the gene expressions of cgy-2- NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF).

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: To study this phenomenon, we analyzed a set of eight poliovirus samples by both pyrosequencing and Illumina methods. .. To study this phenomenon, we analyzed a set of eight poliovirus samples by both pyrosequencing and Illumina methods.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: To study this phenomenon, we analyzed a set of eight poliovirus samples by both pyrosequencing and Illumina methods. .. Various characteristics of both datasets shown in demonstrate that the average sequence length was significantly higher for pyrosequencing (195 vs. 38), but the Illumina method produced roughly 60 times more sequence information. .. Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Various characteristics of both datasets shown in demonstrate that the average sequence length was significantly higher for pyrosequencing (195 vs. 38), but the Illumina method produced roughly 60 times more sequence information. .. Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina.

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Two versions of MPS were used in this study: pyrosequencing (454 Life Sciences, a Roche company) and Illumina methods. .. Whereas pyrosequencing produced longer sequences, the Illumina approach generated roughly 60 times more sequence information, allowing more sensitive detection of minority sequence variants. .. Although the accuracy of base calls was roughly similar, pyrosequencing erroneously identified a significant number of insertions and deletions, especially in regions where the same base is repeated several times.

Article Title: Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Article Snippet: For the most complex community, where there was low coverage of each genome, assemblies from Illumina and pyrosequencing failed to represent the expected functional composition of the metagenomes, as there were very few complete genes annotated. .. For the most complex community, where there was low coverage of each genome, assemblies from Illumina and pyrosequencing failed to represent the expected functional composition of the metagenomes, as there were very few complete genes annotated.

Activation Assay:

Article Title: microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition
Article Snippet: To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis. .. To identify the cellular pathways modulated by miR-181a and define specific targets driving the observed EMT phenotype, we performed high-throughput RNA sequencing (Illumina) on A2780 pBABE and p181a cell lines with a combination of computational target prediction, global transcriptome- and pathway-based analysis.

Marker:

Article Title: Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation
Article Snippet: Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max ) near-isogenic line (cgy-2- NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. .. To identify α-null-related transcriptional changes, the gene expressions of cgy-2- NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF).

Article Title: Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines
Article Snippet: Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina. .. Therefore, the average number of times each nucleotide was sequenced was 885 for pyrosequencing and 56,650 for Illumina.

Variant Assay:

Article Title: Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials
Article Snippet: These results are consistent with our visual observations of DNA quality as well as with the decrease in sequence quality obtained and illustrate that irradiated material can be detected via PCR, yet the sensitivity is sacrificed ( , ). .. In contrast to the requirements for longer DNA fragments during 454 pyrosequencing and Illumina library preparation, qPCR detection schemes typically amplify fragments of less than 140 bp (131 bp for B. atrophaeus variant globigii and 67 bp for Y. pestis in this study). .. The probability of occurrence of radiation damage in any given fragment increases with the strand length; therefore, it is not surprising that methods that are dependent on smaller fragments would be less affected by irradiation than those that require longer strands.

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    Illumina Inc deep illumina sequencing
    Amplicon length distributions of MCP gene fragments from various samples with clone (A) , shallow <t>Illumina</t> sequencing (B) , and deep Illumina sequencing (C) methods. “R1” and “R2” for some samples refer to two sequencing replicates with different barcodes. Three bar-graphs share the same legends. The Y-axis is relative proportion of each length fragment.
    Deep Illumina Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The schematic model for <t>LIF-mediated</t> cancer dissemination in <t>NPC</t>
    Lif Deep Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc mirna deep sequencing
    Network of signal pathways and their respective dysregulated <t>miRNAs</t> in DFs. The red squares represent the upregulated miRNAs while the green squares represent downregulated ones. The purple nodes represent pathways affected by two or three dysregulated miRNAs and the yellow nodes represent pathways coregulated by at least 4 dysregulated miRNAs. Other pale blue circles represent pathways affected by only one differentially expressed <t>miRNA</t> in DFs comparing with SFs.
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    Illumina Inc small rna deep sequencing libraries
    Genetic requirements for primary and secondary siRNA accumulation in L2 transgenic plants. Blot hybridization analysis of total <t>RNA</t> isolated from L2, L2 x rdr6 and L2 x dcl4 plants infected with <t>CaLCuV::GFP</t> viruses Lead, CodM and Trail . The blot was successively hybridized with short DNA probes specific for CaLCuV genes AC4 (AC4_s and AC4_as) and AV1 (A1063_s and A1063_as), 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses ( Lead, CodM, Trail ), GFP mRNA 3′UTR non-target sequence (3′UTR_s) and Arabidopsis miR173 and Met-tRNA (the latter two serve as loading control).
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    Image Search Results


    Amplicon length distributions of MCP gene fragments from various samples with clone (A) , shallow Illumina sequencing (B) , and deep Illumina sequencing (C) methods. “R1” and “R2” for some samples refer to two sequencing replicates with different barcodes. Three bar-graphs share the same legends. The Y-axis is relative proportion of each length fragment.

    Journal: Frontiers in Microbiology

    Article Title: High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

    doi: 10.3389/fmicb.2018.00887

    Figure Lengend Snippet: Amplicon length distributions of MCP gene fragments from various samples with clone (A) , shallow Illumina sequencing (B) , and deep Illumina sequencing (C) methods. “R1” and “R2” for some samples refer to two sequencing replicates with different barcodes. Three bar-graphs share the same legends. The Y-axis is relative proportion of each length fragment.

    Article Snippet: For example, most OTUs from a combined clone library for all samples were detected by both shallow and deep Illumina sequencing methods, and most OTUs from shallow Illumina sequencing were also detected by deep Illumina sequencing ( Figure ).

    Techniques: Amplification, Sequencing

    Rarefaction curves by plotting observed OTU number against sampled sequence number. (A) Clone sequence, (B) shallow Illumina sequence, and (C) deep Illumina sequence.

    Journal: Frontiers in Microbiology

    Article Title: High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

    doi: 10.3389/fmicb.2018.00887

    Figure Lengend Snippet: Rarefaction curves by plotting observed OTU number against sampled sequence number. (A) Clone sequence, (B) shallow Illumina sequence, and (C) deep Illumina sequence.

    Article Snippet: For example, most OTUs from a combined clone library for all samples were detected by both shallow and deep Illumina sequencing methods, and most OTUs from shallow Illumina sequencing were also detected by deep Illumina sequencing ( Figure ).

    Techniques: Sequencing

    UPGMA cluster trees based on unweighted UniFrac distances. The numbers on the nodes refer to the support percentages by 1,000 jackknife tests. UPGMA cluster trees in (A–C) were constructed based on clone sequences, shallow Illumina sequences, and deep Illumina sequences, respectively. “_R1” and “_R2” in sample ID refer to two sequencing replicates with different barcodes.

    Journal: Frontiers in Microbiology

    Article Title: High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

    doi: 10.3389/fmicb.2018.00887

    Figure Lengend Snippet: UPGMA cluster trees based on unweighted UniFrac distances. The numbers on the nodes refer to the support percentages by 1,000 jackknife tests. UPGMA cluster trees in (A–C) were constructed based on clone sequences, shallow Illumina sequences, and deep Illumina sequences, respectively. “_R1” and “_R2” in sample ID refer to two sequencing replicates with different barcodes.

    Article Snippet: For example, most OTUs from a combined clone library for all samples were detected by both shallow and deep Illumina sequencing methods, and most OTUs from shallow Illumina sequencing were also detected by deep Illumina sequencing ( Figure ).

    Techniques: Construct, Sequencing

    Distribution of dominant, full-length MCP gene OTUs (relative abundances higher than 2%) in eight samples. These sequences were obtained based on deep Illumina sequencing.

    Journal: Frontiers in Microbiology

    Article Title: High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

    doi: 10.3389/fmicb.2018.00887

    Figure Lengend Snippet: Distribution of dominant, full-length MCP gene OTUs (relative abundances higher than 2%) in eight samples. These sequences were obtained based on deep Illumina sequencing.

    Article Snippet: For example, most OTUs from a combined clone library for all samples were detected by both shallow and deep Illumina sequencing methods, and most OTUs from shallow Illumina sequencing were also detected by deep Illumina sequencing ( Figure ).

    Techniques: Sequencing

    The schematic model for LIF-mediated cancer dissemination in NPC

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: The schematic model for LIF-mediated cancer dissemination in NPC

    Article Snippet: To determine whether the genetic alterations of LIF were present in NPC, we conducted LIF deep-sequencing (Illumina) on 50 formalin-fixed paraffin-embedded (FFPE) NPC samples as well as Sanger sequencing on 107 FFPE NPC samples (see ) and identified a variety of single nucleotide polymorphism (Fig. ).

    Techniques:

    LIF regulates focal adhesion molecules. a Detection of endogenous focal adhesion kinases in cancer cells via western blot using GAPDH as a loading control. b LIF regulates the spatial distribution of activated focal adhesion kinases. Cells were labeled with antibodies against phospho-PXN (Y118) or phospho-FAK (Y397). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm. c Live imaging of focal adhesion during transendothelial invasion. Cancer cells expressing LifeAct-RFP were pre-labeled with Talin-GFP and plated onto the HUVEC layer. Images were captured 24 h post-plating. Blue, nuclei labeled with Hoechst33342. Scale bars, 20 μm. The white arrow indicates the damaged area caused by Talin-rich elongated protrusion. d Representative images of LIF, p-PXN (Y118), and p-FAK (Y397) expression in paraffin-embedded consecutive NPC tissue sections. Scale bars, 50 μm. e , f Correlation analyses based on IHC scores (Spearman’s correlation test). Correlations were evident between LIF and p-FAK (Y397) ( e ) and LIF and p-PXN (Y118) ( f )

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: LIF regulates focal adhesion molecules. a Detection of endogenous focal adhesion kinases in cancer cells via western blot using GAPDH as a loading control. b LIF regulates the spatial distribution of activated focal adhesion kinases. Cells were labeled with antibodies against phospho-PXN (Y118) or phospho-FAK (Y397). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm. c Live imaging of focal adhesion during transendothelial invasion. Cancer cells expressing LifeAct-RFP were pre-labeled with Talin-GFP and plated onto the HUVEC layer. Images were captured 24 h post-plating. Blue, nuclei labeled with Hoechst33342. Scale bars, 20 μm. The white arrow indicates the damaged area caused by Talin-rich elongated protrusion. d Representative images of LIF, p-PXN (Y118), and p-FAK (Y397) expression in paraffin-embedded consecutive NPC tissue sections. Scale bars, 50 μm. e , f Correlation analyses based on IHC scores (Spearman’s correlation test). Correlations were evident between LIF and p-FAK (Y397) ( e ) and LIF and p-PXN (Y118) ( f )

    Article Snippet: To determine whether the genetic alterations of LIF were present in NPC, we conducted LIF deep-sequencing (Illumina) on 50 formalin-fixed paraffin-embedded (FFPE) NPC samples as well as Sanger sequencing on 107 FFPE NPC samples (see ) and identified a variety of single nucleotide polymorphism (Fig. ).

    Techniques: Western Blot, Labeling, Staining, Imaging, Expressing, Immunohistochemistry

    LIFR–YAP1 signaling is critical for LIF-mediated invasion of NPC cells. a Endogenous protein expression of LIFR, p-YAP1(S127), and YAP1 in three cancer cell lines. b Immunostaining for YAP1 and LIFR in three cancer cell lines. Scale bars, 10 μm. c Western blot analysis of LIFR and p-YAP1 (S127) protein levels in WT and cLIF cancer cells transfected with SMARTpool LIFR siRNA or control siRNA. The p-YAP1 (S127) expressions with respect to total YAP1 levels were quantified and presented as mean ± SEM ( n = 3). At least three independent experiments were performed. d Western blot analysis of expression of focal adhesion molecules and SRC in WT and LIF +/− cancer cells transfected with YAP1 siRNA. GAPDH was used as the loading control. e Representative images for p-PXN (Y118) expression in WT or LIF +/− cancer cells transfected with YAP1 or control siRNA ( n = 3). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Scale bars, 10 μm. f Representative images of the HUVEC layer replacement assay. Fixed numbers of WT or LIF +/− cancer cells transfected with YAP1 or control siRNA were plated onto the confluent HUVEC layer and co-cultivated for 24 h. Cancer cells were labeled with antibody against pan-cytokeratin (red) and HUVEC cells with antibody against VE-cadherin (green). Scale bars, 20 μm. g , h Quantification of displaced areas depicted in f . WT cancer cells ( g ). LIF +/− cancer cells ( h ). Invaded areas were calculated using CellSens imaging software (Olympus). Data are presented with scatter dot plot (mean ± SEM). Each black dot represents one captured image. Mann–Whitney test. i Immunohistochemistry for LIFR and YAP1 expression (brown) in consecutive NPC biopsy sections derived from primary or bone marrow metastatic lesions

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: LIFR–YAP1 signaling is critical for LIF-mediated invasion of NPC cells. a Endogenous protein expression of LIFR, p-YAP1(S127), and YAP1 in three cancer cell lines. b Immunostaining for YAP1 and LIFR in three cancer cell lines. Scale bars, 10 μm. c Western blot analysis of LIFR and p-YAP1 (S127) protein levels in WT and cLIF cancer cells transfected with SMARTpool LIFR siRNA or control siRNA. The p-YAP1 (S127) expressions with respect to total YAP1 levels were quantified and presented as mean ± SEM ( n = 3). At least three independent experiments were performed. d Western blot analysis of expression of focal adhesion molecules and SRC in WT and LIF +/− cancer cells transfected with YAP1 siRNA. GAPDH was used as the loading control. e Representative images for p-PXN (Y118) expression in WT or LIF +/− cancer cells transfected with YAP1 or control siRNA ( n = 3). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Scale bars, 10 μm. f Representative images of the HUVEC layer replacement assay. Fixed numbers of WT or LIF +/− cancer cells transfected with YAP1 or control siRNA were plated onto the confluent HUVEC layer and co-cultivated for 24 h. Cancer cells were labeled with antibody against pan-cytokeratin (red) and HUVEC cells with antibody against VE-cadherin (green). Scale bars, 20 μm. g , h Quantification of displaced areas depicted in f . WT cancer cells ( g ). LIF +/− cancer cells ( h ). Invaded areas were calculated using CellSens imaging software (Olympus). Data are presented with scatter dot plot (mean ± SEM). Each black dot represents one captured image. Mann–Whitney test. i Immunohistochemistry for LIFR and YAP1 expression (brown) in consecutive NPC biopsy sections derived from primary or bone marrow metastatic lesions

    Article Snippet: To determine whether the genetic alterations of LIF were present in NPC, we conducted LIF deep-sequencing (Illumina) on 50 formalin-fixed paraffin-embedded (FFPE) NPC samples as well as Sanger sequencing on 107 FFPE NPC samples (see ) and identified a variety of single nucleotide polymorphism (Fig. ).

    Techniques: Expressing, Immunostaining, Western Blot, Transfection, Staining, Labeling, Imaging, Software, MANN-WHITNEY, Immunohistochemistry, Derivative Assay

    AZD0530 treatment suppresses LIF-mediated tumor invasion. a Western blot analysis of YAP1 and focal adhesion proteins in cancer cells treated with AZD0530 (5 μM). Protein lysates were harvested at 24 h post treatment. b Immunostaining for YAP1 (red) in cancer cells treated with AZD0530. Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm. c – e Representative images of YAP1 ( c ), p-PXN (Y118) ( d ), and p-FAK (Y397) ( e ) expression in mouse WT and cLIF xenografts treated with AZD0530 or vehicle. Scale bars, 50 μm. f Quantification of mouse NPC xenografts with events of local invasion based on results of hematoxylin and eosin staining. The AZD0530 treatment procedure in the mouse model is described in Methods

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: AZD0530 treatment suppresses LIF-mediated tumor invasion. a Western blot analysis of YAP1 and focal adhesion proteins in cancer cells treated with AZD0530 (5 μM). Protein lysates were harvested at 24 h post treatment. b Immunostaining for YAP1 (red) in cancer cells treated with AZD0530. Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm. c – e Representative images of YAP1 ( c ), p-PXN (Y118) ( d ), and p-FAK (Y397) ( e ) expression in mouse WT and cLIF xenografts treated with AZD0530 or vehicle. Scale bars, 50 μm. f Quantification of mouse NPC xenografts with events of local invasion based on results of hematoxylin and eosin staining. The AZD0530 treatment procedure in the mouse model is described in Methods

    Article Snippet: To determine whether the genetic alterations of LIF were present in NPC, we conducted LIF deep-sequencing (Illumina) on 50 formalin-fixed paraffin-embedded (FFPE) NPC samples as well as Sanger sequencing on 107 FFPE NPC samples (see ) and identified a variety of single nucleotide polymorphism (Fig. ).

    Techniques: Western Blot, Immunostaining, Staining, Expressing

    Elevated cytoplasmic LIF and LIFR in NPC are correlated with poorer prognosis. a Representative images of LIF expression in adjacent normal epithelium and NPC tumor tissues. Scale bars, 20 μm. b Statistical analysis of cytoplasmic LIF expression in primary NPC tumor tissues and metastatic lesions. Analysis of cytoplasmic LIF expression in distinct metastatic lesions is shown (right). ** p

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: Elevated cytoplasmic LIF and LIFR in NPC are correlated with poorer prognosis. a Representative images of LIF expression in adjacent normal epithelium and NPC tumor tissues. Scale bars, 20 μm. b Statistical analysis of cytoplasmic LIF expression in primary NPC tumor tissues and metastatic lesions. Analysis of cytoplasmic LIF expression in distinct metastatic lesions is shown (right). ** p

    Article Snippet: To determine whether the genetic alterations of LIF were present in NPC, we conducted LIF deep-sequencing (Illumina) on 50 formalin-fixed paraffin-embedded (FFPE) NPC samples as well as Sanger sequencing on 107 FFPE NPC samples (see ) and identified a variety of single nucleotide polymorphism (Fig. ).

    Techniques: Expressing

    Characterization of LIF mutant clones. a Sequence analysis of the LIF gene. Genomic DNA was extracted from parental NPC BM1 cells with wild-type LIF or established clones either with mutations in the signal peptide region of LIF (cLIF clone) or loss of the initiating codon in one allele (LIF +/− clone). The initiating codon within the spacer is indicated in red. Mutated nucleotides are marked in blue. b Assessment of LIF protein expression via western blot using GAPDH as a loading control. c Assessment of secreted LIF using a bead-based cytokine assay. Supernatants were harvested 2 days post culture. Data are presented as means ± SD of triplicate experiments. ** p

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: Characterization of LIF mutant clones. a Sequence analysis of the LIF gene. Genomic DNA was extracted from parental NPC BM1 cells with wild-type LIF or established clones either with mutations in the signal peptide region of LIF (cLIF clone) or loss of the initiating codon in one allele (LIF +/− clone). The initiating codon within the spacer is indicated in red. Mutated nucleotides are marked in blue. b Assessment of LIF protein expression via western blot using GAPDH as a loading control. c Assessment of secreted LIF using a bead-based cytokine assay. Supernatants were harvested 2 days post culture. Data are presented as means ± SD of triplicate experiments. ** p

    Article Snippet: To determine whether the genetic alterations of LIF were present in NPC, we conducted LIF deep-sequencing (Illumina) on 50 formalin-fixed paraffin-embedded (FFPE) NPC samples as well as Sanger sequencing on 107 FFPE NPC samples (see ) and identified a variety of single nucleotide polymorphism (Fig. ).

    Techniques: Mutagenesis, Clone Assay, Sequencing, Expressing, Western Blot, Cytokine Assay

    Network of signal pathways and their respective dysregulated miRNAs in DFs. The red squares represent the upregulated miRNAs while the green squares represent downregulated ones. The purple nodes represent pathways affected by two or three dysregulated miRNAs and the yellow nodes represent pathways coregulated by at least 4 dysregulated miRNAs. Other pale blue circles represent pathways affected by only one differentially expressed miRNA in DFs comparing with SFs.

    Journal: BioMed Research International

    Article Title: MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

    doi: 10.1155/2017/4585213

    Figure Lengend Snippet: Network of signal pathways and their respective dysregulated miRNAs in DFs. The red squares represent the upregulated miRNAs while the green squares represent downregulated ones. The purple nodes represent pathways affected by two or three dysregulated miRNAs and the yellow nodes represent pathways coregulated by at least 4 dysregulated miRNAs. Other pale blue circles represent pathways affected by only one differentially expressed miRNA in DFs comparing with SFs.

    Article Snippet: To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing.

    Techniques:

    TGF- β signaling pathway. This map of TGF- β signaling pathway was based on KEGG where the red boxes represent target genes regulated by significantly differentially expressed miRNAs through predicting results.

    Journal: BioMed Research International

    Article Title: MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

    doi: 10.1155/2017/4585213

    Figure Lengend Snippet: TGF- β signaling pathway. This map of TGF- β signaling pathway was based on KEGG where the red boxes represent target genes regulated by significantly differentially expressed miRNAs through predicting results.

    Article Snippet: To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing.

    Techniques:

    Network of abundantly expressed miRNAs and correlative pathways in both DFs and SFs. Yellow nodes represent abundantly expressed miRNAs in common while blue nodes represent the pathways affected by miRNAs.

    Journal: BioMed Research International

    Article Title: MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

    doi: 10.1155/2017/4585213

    Figure Lengend Snippet: Network of abundantly expressed miRNAs and correlative pathways in both DFs and SFs. Yellow nodes represent abundantly expressed miRNAs in common while blue nodes represent the pathways affected by miRNAs.

    Article Snippet: To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing.

    Techniques:

    Network of SMAD2 and relevant pathways regulated by identified miRNAs. The yellow circular nodes represent highly abundant miRNAs both in DFs and SFs. Red circular nodes represent the upregulated miRNAs in DFs and the green circular nodes represent the downregulated miRNAs in DFs. The red and green triangular nodes represent up- and downregulated miRNAs in DFs when compared with either small healthy follicles or SFs, respectively. The purple square nodes represent pathways regulated by these identified miRNAs and SMAD2 simultaneously.

    Journal: BioMed Research International

    Article Title: MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

    doi: 10.1155/2017/4585213

    Figure Lengend Snippet: Network of SMAD2 and relevant pathways regulated by identified miRNAs. The yellow circular nodes represent highly abundant miRNAs both in DFs and SFs. Red circular nodes represent the upregulated miRNAs in DFs and the green circular nodes represent the downregulated miRNAs in DFs. The red and green triangular nodes represent up- and downregulated miRNAs in DFs when compared with either small healthy follicles or SFs, respectively. The purple square nodes represent pathways regulated by these identified miRNAs and SMAD2 simultaneously.

    Article Snippet: To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing.

    Techniques:

    MAPK signaling pathway. This map of MAPK signaling pathway was obtained based on KEGG. The yellow boxes represent target genes which were regulated by the 7 critical miRNAs identified by previous analysis.

    Journal: BioMed Research International

    Article Title: MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

    doi: 10.1155/2017/4585213

    Figure Lengend Snippet: MAPK signaling pathway. This map of MAPK signaling pathway was obtained based on KEGG. The yellow boxes represent target genes which were regulated by the 7 critical miRNAs identified by previous analysis.

    Article Snippet: To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing.

    Techniques:

    Network of the common differentially expressed miRNAs and their functionally associated signaling pathways in DFs comparing with both small healthy follicles and SFs. The red squares represent the upregulated miRNAs while green squares represent downregulated ones. The purple nodes represent pathways affected by two or three dysregulated miRNAs and the yellow nodes represent pathways coregulated by at least 4 dysregulated miRNAs. Other pale blue circles represent pathways affected by only one differentially expressed miRNA in DFs comparing with both small healthy follicles and SFs.

    Journal: BioMed Research International

    Article Title: MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

    doi: 10.1155/2017/4585213

    Figure Lengend Snippet: Network of the common differentially expressed miRNAs and their functionally associated signaling pathways in DFs comparing with both small healthy follicles and SFs. The red squares represent the upregulated miRNAs while green squares represent downregulated ones. The purple nodes represent pathways affected by two or three dysregulated miRNAs and the yellow nodes represent pathways coregulated by at least 4 dysregulated miRNAs. Other pale blue circles represent pathways affected by only one differentially expressed miRNA in DFs comparing with both small healthy follicles and SFs.

    Article Snippet: To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing.

    Techniques:

    Network of miRNAs and TFs. (a) Network of TFs and pivotal miRNAs confirmed in comparison of DFs and SFs. (b) The core network shrank by extracting the hub TFs from (a). The red nodes represent abundantly expressed miRNAs both in DFs and SFs. The yellow nodes represent upregulated and the green nodes represent downregulated miRNAs. The pink rhombic nodes represent TFs affected by differentially expressed miRNAs. (c) Network of TFs and critical miRNAs confirmed in comparison of DFs versus SFs as well as DFs versus small follicles. (d) The relevant core network constructed by the relations between the hub TFs and miRNAs with correspondence to (c). The red nodes represent upregulated miRNAs and green nodes represent downregulated miRNAs. The purple rhombic nodes represent TFs affected by both differentially expressed miRNAs. The orange lines display the connections of TFs and miRNA which had evidenced basis [ 43 – 46 ].

    Journal: BioMed Research International

    Article Title: MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

    doi: 10.1155/2017/4585213

    Figure Lengend Snippet: Network of miRNAs and TFs. (a) Network of TFs and pivotal miRNAs confirmed in comparison of DFs and SFs. (b) The core network shrank by extracting the hub TFs from (a). The red nodes represent abundantly expressed miRNAs both in DFs and SFs. The yellow nodes represent upregulated and the green nodes represent downregulated miRNAs. The pink rhombic nodes represent TFs affected by differentially expressed miRNAs. (c) Network of TFs and critical miRNAs confirmed in comparison of DFs versus SFs as well as DFs versus small follicles. (d) The relevant core network constructed by the relations between the hub TFs and miRNAs with correspondence to (c). The red nodes represent upregulated miRNAs and green nodes represent downregulated miRNAs. The purple rhombic nodes represent TFs affected by both differentially expressed miRNAs. The orange lines display the connections of TFs and miRNA which had evidenced basis [ 43 – 46 ].

    Article Snippet: To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing.

    Techniques: Construct

    Genetic requirements for primary and secondary siRNA accumulation in L2 transgenic plants. Blot hybridization analysis of total RNA isolated from L2, L2 x rdr6 and L2 x dcl4 plants infected with CaLCuV::GFP viruses Lead, CodM and Trail . The blot was successively hybridized with short DNA probes specific for CaLCuV genes AC4 (AC4_s and AC4_as) and AV1 (A1063_s and A1063_as), 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses ( Lead, CodM, Trail ), GFP mRNA 3′UTR non-target sequence (3′UTR_s) and Arabidopsis miR173 and Met-tRNA (the latter two serve as loading control).

    Journal: PLoS Pathogens

    Article Title: Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

    doi: 10.1371/journal.ppat.1002941

    Figure Lengend Snippet: Genetic requirements for primary and secondary siRNA accumulation in L2 transgenic plants. Blot hybridization analysis of total RNA isolated from L2, L2 x rdr6 and L2 x dcl4 plants infected with CaLCuV::GFP viruses Lead, CodM and Trail . The blot was successively hybridized with short DNA probes specific for CaLCuV genes AC4 (AC4_s and AC4_as) and AV1 (A1063_s and A1063_as), 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses ( Lead, CodM, Trail ), GFP mRNA 3′UTR non-target sequence (3′UTR_s) and Arabidopsis miR173 and Met-tRNA (the latter two serve as loading control).

    Article Snippet: Table S1 Counts of viral and endogenous small RNAs in the Illumina small RNA deep-sequencing libraries for mock-inoculated and wild type CaLCuV-infected Col-0 and rdr1/2/6 plants (S1A), mock inoculated and CaLCuV::Chl-infected Col-0 plants (S1B), mock inoculated and CaLCuV:: GFP-Pro-FL -infected Col-0 plants (S1C), mock inoculated and CaLCuV:: GFP-Enh -infected Col-0 plants (S1D), mock inoculated and CaLCuV:: GFP-Core -infected Col-0 plants (S1E), mock inoculated and CaLCuV:: GFP-Lead -infected Col-0 plants (S1F), mock inoculated and CaLCuV:: GFP-CodM -infected Col-0 plants (S1G), mock inoculated and CaLCuV:: GFP-Trail -infected Col-0 plants (S1H), and mock inoculated and CaLCuV:: GFP-PolyA -infected Col-0 plants (S1I). (XLSX) Click here for additional data file.

    Techniques: Transgenic Assay, Hybridization, Isolation, Infection, Sequencing

    VIGS phenotypes and primary siRNA accumulation in L2 transgenic plants infected with CaLCuV::GFP viruses that target the GFP promoter and terminator elements. ( A ) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer ( Enh ) and core promoter ( Core ) elements (CAAT and TATA boxes and transcription start Plus1 ), the GFP mRNA elements (5′UTR, AUG and UAA codons and 3′UTR, and 35S terminator are indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses ProFL, Enh, CAAT, TATA, Plus1, CodFL, Trail and Post are indicated with dotted boxes; ( B ) and ( C ) Blot hybridization analysis of total RNA isolated from L2 plants infected with the above viruses. The two blots were successively hybridized with short DNA probes specific for CaLCuV AC4 gene (AC4_s) and the 35S::GFP transgene sequences inserted in CaLCuV::GFP viruses and Arabidopsis miR173 and Met-tRNA (the latter two serve as loading control). ( D ) Pictures under UV light of L2 transgenic plans infected with the CaLCuV::GFP viruses (names indicated).

    Journal: PLoS Pathogens

    Article Title: Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

    doi: 10.1371/journal.ppat.1002941

    Figure Lengend Snippet: VIGS phenotypes and primary siRNA accumulation in L2 transgenic plants infected with CaLCuV::GFP viruses that target the GFP promoter and terminator elements. ( A ) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer ( Enh ) and core promoter ( Core ) elements (CAAT and TATA boxes and transcription start Plus1 ), the GFP mRNA elements (5′UTR, AUG and UAA codons and 3′UTR, and 35S terminator are indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses ProFL, Enh, CAAT, TATA, Plus1, CodFL, Trail and Post are indicated with dotted boxes; ( B ) and ( C ) Blot hybridization analysis of total RNA isolated from L2 plants infected with the above viruses. The two blots were successively hybridized with short DNA probes specific for CaLCuV AC4 gene (AC4_s) and the 35S::GFP transgene sequences inserted in CaLCuV::GFP viruses and Arabidopsis miR173 and Met-tRNA (the latter two serve as loading control). ( D ) Pictures under UV light of L2 transgenic plans infected with the CaLCuV::GFP viruses (names indicated).

    Article Snippet: Table S1 Counts of viral and endogenous small RNAs in the Illumina small RNA deep-sequencing libraries for mock-inoculated and wild type CaLCuV-infected Col-0 and rdr1/2/6 plants (S1A), mock inoculated and CaLCuV::Chl-infected Col-0 plants (S1B), mock inoculated and CaLCuV:: GFP-Pro-FL -infected Col-0 plants (S1C), mock inoculated and CaLCuV:: GFP-Enh -infected Col-0 plants (S1D), mock inoculated and CaLCuV:: GFP-Core -infected Col-0 plants (S1E), mock inoculated and CaLCuV:: GFP-Lead -infected Col-0 plants (S1F), mock inoculated and CaLCuV:: GFP-CodM -infected Col-0 plants (S1G), mock inoculated and CaLCuV:: GFP-Trail -infected Col-0 plants (S1H), and mock inoculated and CaLCuV:: GFP-PolyA -infected Col-0 plants (S1I). (XLSX) Click here for additional data file.

    Techniques: Transgenic Assay, Infection, Hybridization, Isolation

    VIGS phenotypes and accumulation of primary and secondary siRNAs in L2 transgenic plants infected with CaLCuV::GFP viruses targeting the GFP transcribed region. ( A ) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer and core promoter elements, GFP mRNA elements including 5′UTR, translation start (AUG) and stop (UAA) codons and 3′UTR with poly(A) signal (AAUAAA), and 35S terminator sequences indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses Lead , CodB, CodM, CodE, Trail and polyA , are indicated with dotted boxes; ( B ) Pictures under UV light of L2 transgenic plants infected with the above viruses; ( C ) Blot hybridization analysis of total RNA isolated from plants shown in Panel B. The blot was successively hybridized with short DNA probes specific for CaLCuV AC4 gene (AC4_s) and 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses ( Lead, CodB, CodM, CodE, Trail and polyA ), the GFP mRNA 3′UTR non-target sequence (3′UTR) and Arabidopsis miR173 and Met-tRNA (the latter two serve as loading control).

    Journal: PLoS Pathogens

    Article Title: Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

    doi: 10.1371/journal.ppat.1002941

    Figure Lengend Snippet: VIGS phenotypes and accumulation of primary and secondary siRNAs in L2 transgenic plants infected with CaLCuV::GFP viruses targeting the GFP transcribed region. ( A ) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer and core promoter elements, GFP mRNA elements including 5′UTR, translation start (AUG) and stop (UAA) codons and 3′UTR with poly(A) signal (AAUAAA), and 35S terminator sequences indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses Lead , CodB, CodM, CodE, Trail and polyA , are indicated with dotted boxes; ( B ) Pictures under UV light of L2 transgenic plants infected with the above viruses; ( C ) Blot hybridization analysis of total RNA isolated from plants shown in Panel B. The blot was successively hybridized with short DNA probes specific for CaLCuV AC4 gene (AC4_s) and 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses ( Lead, CodB, CodM, CodE, Trail and polyA ), the GFP mRNA 3′UTR non-target sequence (3′UTR) and Arabidopsis miR173 and Met-tRNA (the latter two serve as loading control).

    Article Snippet: Table S1 Counts of viral and endogenous small RNAs in the Illumina small RNA deep-sequencing libraries for mock-inoculated and wild type CaLCuV-infected Col-0 and rdr1/2/6 plants (S1A), mock inoculated and CaLCuV::Chl-infected Col-0 plants (S1B), mock inoculated and CaLCuV:: GFP-Pro-FL -infected Col-0 plants (S1C), mock inoculated and CaLCuV:: GFP-Enh -infected Col-0 plants (S1D), mock inoculated and CaLCuV:: GFP-Core -infected Col-0 plants (S1E), mock inoculated and CaLCuV:: GFP-Lead -infected Col-0 plants (S1F), mock inoculated and CaLCuV:: GFP-CodM -infected Col-0 plants (S1G), mock inoculated and CaLCuV:: GFP-Trail -infected Col-0 plants (S1H), and mock inoculated and CaLCuV:: GFP-PolyA -infected Col-0 plants (S1I). (XLSX) Click here for additional data file.

    Techniques: Transgenic Assay, Infection, Hybridization, Isolation, Sequencing

    Accumulation of long viral nucleic acids and vsRNAs in wild type versus rdr1/2/6 triple mutant plants. Total RNA and total DNA from CaLCuV-infected Arabidopsis wt (Col-0) and rdr1/2/6 plants was analyzed by RNA blot hybridization using 5% ( A ) and 15% ( B ) PAGE and by Southern blot hybridization ( C ). The RNA blot membranes were successively hybridized with mixtures of DNA oligonucleotide probes complementary to respective viral mRNAs (for sequences, see Protocol S1 ) and, in the case of sRNA analysis, single DNA oligonucleotide probes specific for vsRNA of sense or antisense polarity and the endogenous Arabidopsis miRNA (22 nt miR173), tasiRNA (21 nt siR255) and hcsiRNA (24 nt siR1003). The Southern blot membranes were hybridized with long dsDNA probes specific for DNA-A or DNA-B. Positions of co-migrating forms of viral DNA including open-circular double-stranded (dsDNA), supercoiled (scDNA) and single-stranded (ssDNA) are indicated by arrows; the smear of shorter (than monomeric) ssDNA is also indicated. EtBr staining of total RNA ( A ) or plant genomic DNA ( C ) is shown as loading control. The size markers are indicated on each scan. Positions of viral mRNAs are indicated by asterisks. ( D ) Real time qPCR measurement of relative accumulation of viral polyadenylated mRNAs (left) and total viral DNAs A and B (right) in wild type versus rdr/1/2/6 mutant plants. For each mRNA and each DNA, the accumulation level in the wild type sample is set to 1.

    Journal: PLoS Pathogens

    Article Title: Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

    doi: 10.1371/journal.ppat.1002941

    Figure Lengend Snippet: Accumulation of long viral nucleic acids and vsRNAs in wild type versus rdr1/2/6 triple mutant plants. Total RNA and total DNA from CaLCuV-infected Arabidopsis wt (Col-0) and rdr1/2/6 plants was analyzed by RNA blot hybridization using 5% ( A ) and 15% ( B ) PAGE and by Southern blot hybridization ( C ). The RNA blot membranes were successively hybridized with mixtures of DNA oligonucleotide probes complementary to respective viral mRNAs (for sequences, see Protocol S1 ) and, in the case of sRNA analysis, single DNA oligonucleotide probes specific for vsRNA of sense or antisense polarity and the endogenous Arabidopsis miRNA (22 nt miR173), tasiRNA (21 nt siR255) and hcsiRNA (24 nt siR1003). The Southern blot membranes were hybridized with long dsDNA probes specific for DNA-A or DNA-B. Positions of co-migrating forms of viral DNA including open-circular double-stranded (dsDNA), supercoiled (scDNA) and single-stranded (ssDNA) are indicated by arrows; the smear of shorter (than monomeric) ssDNA is also indicated. EtBr staining of total RNA ( A ) or plant genomic DNA ( C ) is shown as loading control. The size markers are indicated on each scan. Positions of viral mRNAs are indicated by asterisks. ( D ) Real time qPCR measurement of relative accumulation of viral polyadenylated mRNAs (left) and total viral DNAs A and B (right) in wild type versus rdr/1/2/6 mutant plants. For each mRNA and each DNA, the accumulation level in the wild type sample is set to 1.

    Article Snippet: Table S1 Counts of viral and endogenous small RNAs in the Illumina small RNA deep-sequencing libraries for mock-inoculated and wild type CaLCuV-infected Col-0 and rdr1/2/6 plants (S1A), mock inoculated and CaLCuV::Chl-infected Col-0 plants (S1B), mock inoculated and CaLCuV:: GFP-Pro-FL -infected Col-0 plants (S1C), mock inoculated and CaLCuV:: GFP-Enh -infected Col-0 plants (S1D), mock inoculated and CaLCuV:: GFP-Core -infected Col-0 plants (S1E), mock inoculated and CaLCuV:: GFP-Lead -infected Col-0 plants (S1F), mock inoculated and CaLCuV:: GFP-CodM -infected Col-0 plants (S1G), mock inoculated and CaLCuV:: GFP-Trail -infected Col-0 plants (S1H), and mock inoculated and CaLCuV:: GFP-PolyA -infected Col-0 plants (S1I). (XLSX) Click here for additional data file.

    Techniques: Mutagenesis, Infection, Northern blot, Hybridization, Polyacrylamide Gel Electrophoresis, Southern Blot, Staining, Real-time Polymerase Chain Reaction