Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

High-throughput semi-quantitative analysis of insertional mutations in heterogeneous tumors

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Jos Jonkers

PROVIDER: E-GEOD-22657 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors.

Koudijs Marco J MJ Klijn Christiaan C van der Weyden Louise L Kool Jaap J ten Hoeve Jelle J Sie Daoud D Prasetyanti Pramudita R PR Schut Eva E Kas Sjors S Whipp Theodore T Cuppen Edwin E Wessels Lodewyk L Adams David J DJ Jonkers Jos J

Genome research 20110818 12

Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzym ...[more]

PMID: 21852388

Similar Datasets

Project description:Transcription factors direct gene expression, and so there is much interest in mapping their genome-wide binding locations. M-BM- Current methods do not allow for the multiplexed analysis of TF binding, and this limits their throughput. We describe a novel method for determining the genomic target genes of multiple transcription factors simultaneously. DNA-binding proteins are endowed with the ability to direct transposon insertions into the genome near to where they bind. The transposon becomes a M-bM-^@M-^\Calling CardM-bM-^@M-^] marking the visit of the DNA-binding protein to that location. A unique sequence M-bM-^@M-^\barcodeM-bM-^@M-^] in the transposon matches it to the DNA-binding protein that directed its insertion. The sequences of the DNA flanking the transposon (which reveal where in the genome the transposon landed) and the barcode within the transposon (which identifies the TF that put it there) are determined by massively-parallel DNA sequencing. To demonstrate the methodM-bM-^@M-^Ys feasibility, we determined the genomic targets of eight transcription factors in a single experiment. The Calling Card method promises to significantly reduce the cost and labor needed to determine the genomic targets of many transcription factors in different environmental conditions and genetic backgrounds. These data contain Ty5 insertion sites mapped by an Illumina GAII analyzer in the S. cerevisiae genome for the background strain without any Sir4 present (1 run), in strains expressing Sir4-tagged copies of three well-characterized TFs: Gal4, Leu3, and Gcn4 (1 run each), and a multiplex of eight Sir4-tagged TFs pooled in a single experiment (2 biological replicates), and insertions from the Thi2-Sir4 fusion expressed from its native locus in two conditions (1 run each). The format of each insertions file is [chromosome number] [position of genomic base] [direction of insertion] [number of reads at that position]. Raw sequencing data comes in two varieties. Paired-end data contains a 5 bp barcode at the beginning of read #2. Single-end data contains a 2 bp barcode on the beggining of read #1.

Dataset Information

High-throughput semi-quantitative analysis of insertional mutations in heterogeneous tumors

Publications

High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure