Mammalia
Ontology highlight
ABSTRACT:
PROVIDER: PRJDB10602 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DRR333234.fastq.gz | Fastqsanger.gz | |||
| DRR333235.fastq.gz | Fastqsanger.gz | |||
| DRR333236.fastq.gz | Fastqsanger.gz | |||
| DRR333237.fastq.gz | Fastqsanger.gz | |||
| DRR333238.fastq.gz | Fastqsanger.gz |
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Project description:Exploiting the full potential of insertional mutagenesis screens with retroviruses and transposons requires methods for distinguishing clonal from subclonal insertion events within heterogeneous tumor cell populations. Current protocols, based on ligation mediated PCR, depend on endonuclease based fragmentation of genomic DNA, resulting in strong biases in amplification and sequencing due to a fixed product sizes of the amplicon. We have developed a method called shear-splink, which enables the semi-quantitative high-throughput sequence analysis of insertional mutations, enabling us to count the number of cells harboring a given integration, within a heterogeneous sample. The shear-splink method enriches for (sub)clonal integrations, thereby reducing the contribution of irrelevant passenger mutations normally hampering a reliable identification of common integration sites. Additionally, this improvement allows us to identify genetic interactions between affected genes, co-occurring mutations and to study acquired resistance mechanisms both in vivo and in vitro. Sequencing of retrovrial integration sites by LM-PCR. The associated manuscript describes a new method to quantitatively determine retrovrial integration sites using an improved ligation-mediated PCR approach and subsequent 454 pyrosequencing. [GSM562151 to GSM562159]: Sequence data from different mixtures of 2 different cell lines (called AE6 and BB12) which are processed without a restriction enzyme. These cell lines are derived from an MMTV induced mammary tumor, for which we amplify the MMTV integration sites using a ligation-mediated PCR setup. We mixed these 2 cell lines, both with a different integration spectrum, to determine whether our amplification and sequencing protocol is quantitative, meaning that the coverage per integration site is decreasing upon a further dilution of the sample. [GSM641935 to GSM641950]: Unique Sleeping beauty induced lymphoma specimens (spleen) obtained from a cohort of 16 wild-type mice with the 129P2/C57BL/6J mixed background. [GSM776576 to GSM776956]: The 379 submitted specimens are originating from 127 unique leukemia/lymphoma samples, processed using 3 different techniques in order to identify Sleeping Beauty integration sites. We compared restriction enzyme based LM-PCR (RE-splink) with shearing based LM-PCR (shear-splink) on 127 unique Sleeping Beauty (SB) induced leukemia's/lymphomas. All sequence data generated by the 454 sequencing platform are submitted to GEO, including the final output of our sequence analysis pipeline (in bed format; see Supplementary files linked below). Previous submissions contained similar sequence information (integration sites of viruses or transposons driving tumorigenesis) and are all part of the same manuscript.
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