Ontology highlight
ABSTRACT:
PROVIDER: PRJNA763883 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15914094_1.fastq.gz | Fastqsanger.gz | |||
| SRR15914094_2.fastq.gz | Fastqsanger.gz | |||
| SRR15914095_1.fastq.gz | Fastqsanger.gz | |||
| SRR15914095_2.fastq.gz | Fastqsanger.gz | |||
| SRR15914096_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 16 |
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Project description:Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro, and these sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. Genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal “sweet-spot” GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this was not known. We now demonstrate the absolute necessity of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene, as well as for Ewing sarcoma proliferation and oncogenic transformation. Biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion) demonstrated a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter GGAA-microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the GGAA-microsatellite was increased to the “sweet-spot” length. In contrast, a fully-functional EWS/FLI mutant (Mut9) that retains approximately half of the EWS portion of the fusion showed low affinity for smaller GGAA-microsatellites, but instead significantly increased its affinity at “sweet-spot” microsatellite lengths. Single-gene ChIP and genome-wide ChIP-seq and RNA-seq studies extended these findings to the in vivo setting. Taken together, these data demonstrate the absolute requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unsuspected novel role for the EWS portion of the EWS/FLI fusion in binding to optimal-length GGAA-microsatellites.
2017-10-22 | GSE94480 | GEO
Project description:Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro, and these sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. Genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal “sweet-spot” GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this was not known. We now demonstrate the absolute necessity of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene, as well as for Ewing sarcoma proliferation and oncogenic transformation. Biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion) demonstrated a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter GGAA-microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the GGAA-microsatellite was increased to the “sweet-spot” length. In contrast, a fully-functional EWS/FLI mutant (Mut9) that retains approximately half of the EWS portion of the fusion showed low affinity for smaller GGAA-microsatellites, but instead significantly increased its affinity at “sweet-spot” microsatellite lengths. Single-gene ChIP and genome-wide ChIP-seq and RNA-seq studies extended these findings to the in vivo setting. Taken together, these data demonstrate the absolute requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unsuspected novel role for the EWS portion of the EWS/FLI fusion in binding to optimal-length GGAA-microsatellites.
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Project description:Microsatellites are short tandem repeats (STRs) of a motif of 1 to 6 nucleotides that are ubiquitous in almost all genomes and widely used in many biomedical applications. However, despite the development of next-generation sequencing (NGS) over the past two decades with new technologies coming to the market, accurately sequencing and genotyping STRs, particularly homopolymers, are still very challenging today due to several technical limitations. This leads in many cases to erroneous allele calls and difficulty in correctly identifying the genuine allele distribution in a sample. In the present study, we assessed several second and third NGS approaches in their capability to correctly determine the length of microsatellites using plasmids containing A/T homopolymers, AC/TG or AT/TA dinucleotide STRs of variable length. Standard PCR-free and PCR-containing, single Unique Molecular Index (UMI) and dual UMI ‘duplex sequencing’ protocols were evaluated using Illumina short-read sequencing, and two PCR-free protocols using PacBio and nanopore long-read sequencing. Several bioinformatics algorithms were developed to correctly identify microsatellite alleles from sequencing data, including four and two modes for generating standard and combined consensus alleles, respectively. We provided a detailed analysis and comparison of these approaches and made several recommendations for the accurate determination of microsatellite allele length.
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Project description:Microsatellites are short tandem repeats (STRs) of a motif of 1 to 6 nucleotides that are ubiquitous in almost all genomes and widely used in many biomedical applications. However, despite the development of next-generation sequencing (NGS) over the past two decades with new technologies coming to the market, accurately sequencing and genotyping STRs, particularly homopolymers, are still very challenging today due to several technical limitations. This leads in many cases to erroneous allele calls and difficulty in correctly identifying the genuine allele distribution in a sample. In the present study, we assessed several second and third NGS approaches in their capability to correctly determine the length of microsatellites using plasmids containing A/T homopolymers, AC/TG or AT/TA dinucleotide STRs of variable length. Standard PCR-free and PCR-containing, single Unique Molecular Index (UMI) and dual UMI ‘duplex sequencing’ protocols were evaluated using Illumina short-read sequencing, and two PCR-free protocols using PacBio and nanopore long-read sequencing. Several bioinformatics algorithms were developed to correctly identify microsatellite alleles from sequencing data, including four and two modes for generating standard and combined consensus alleles, respectively. We provided a detailed analysis and comparison of these approaches and made several recommendations for the accurate determination of microsatellite allele length.
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Project description:Microsatellites are short tandem repeats (STRs) of a motif of 1 to 6 nucleotides that are ubiquitous in almost all genomes and widely used in many biomedical applications. However, despite the development of next-generation sequencing (NGS) over the past two decades with new technologies coming to the market, accurately sequencing and genotyping STRs, particularly homopolymers, are still very challenging today due to several technical limitations. This leads in many cases to erroneous allele calls and difficulty in correctly identifying the genuine allele distribution in a sample. In the present study, we assessed several second and third NGS approaches in their capability to correctly determine the length of microsatellites using plasmids containing A/T homopolymers, AC/TG or AT/TA dinucleotide STRs of variable length. Standard PCR-free and PCR-containing, single Unique Molecular Index (UMI) and dual UMI ‘duplex sequencing’ protocols were evaluated using Illumina short-read sequencing, and two PCR-free protocols using PacBio and nanopore long-read sequencing. Several bioinformatics algorithms were developed to correctly identify microsatellite alleles from sequencing data, including four and two modes for generating standard and combined consensus alleles, respectively. We provided a detailed analysis and comparison of these approaches and made several recommendations for the accurate determination of microsatellite allele length.
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Project description:Microsatellites are short tandem repeats (STRs) of a motif of 1 to 6 nucleotides that are ubiquitous in almost all genomes and widely used in many biomedical applications. However, despite the development of next-generation sequencing (NGS) over the past two decades with new technologies coming to the market, accurately sequencing and genotyping STRs, particularly homopolymers, are still very challenging today due to several technical limitations. This leads in many cases to erroneous allele calls and difficulty in correctly identifying the genuine allele distribution in a sample. In the present study, we assessed several second and third NGS approaches in their capability to correctly determine the length of microsatellites using plasmids containing A/T homopolymers, AC/TG or AT/TA dinucleotide STRs of variable length. Standard PCR-free and PCR-containing, single Unique Molecular Index (UMI) and dual UMI ‘duplex sequencing’ protocols were evaluated using Illumina short-read sequencing, and two PCR-free protocols using PacBio and nanopore long-read sequencing. Several bioinformatics algorithms were developed to correctly identify microsatellite alleles from sequencing data, including four and two modes for generating standard and combined consensus alleles, respectively. We provided a detailed analysis and comparison of these approaches and made several recommendations for the accurate determination of microsatellite allele length.
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