Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Improvements in sample preparation for ChIP-chip assays

ABSTRACT: We have improved the new protocol for ChIP-chip by using pooling method. The new method has produced reproducible binding patterns and low background signals. We have performed and compared three methods for ChIP-chip samples: LMPCR, 10 Pooled Samples and WGA.

ORGANISM(S): Homo sapiens

SUBMITTER: Peggy Farnham

PROVIDER: E-GEOD-5251 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Comparison of sample preparation methods for ChIP-chip assays.

O'Geen Henriette H Nicolet Charles M CM Blahnik Kim K Green Roland R Farnham Peggy J PJ

BioTechniques 20061101 5

A single chromatin immunoprecipitation (ChIP) sample does not provide enough DNA for hybridization to a genomic tiling array. A commonly used technique for amplifying the DNA obtained from ChIP assays is ligation-mediated PCR (LM-PCR). However; using this amplification method, we could not identify Oct4 binding sites on genomic tiling arrays representing 1% of the human genome (ENCODE arrays). In contrast, hybridization of a pool of 10 ChIP samples to the arrays produced reproducible binding pat ...[more]

PMID: 17140114

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Project description:The goals of the study were to assess array platform performance and analysis algorithm performance. The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: ChIP-chip, competition, artificial DNA For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf DNA samples had spike-in DNA added to simulate peaks in ChIP-chip experiments. Same DNA samples used in the arrays in this Series; these are technical array replicates.

Project description:The goals of the study were to assess array platform performance and analysis algorithm performance. The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: ChIP-chip, competition For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf DNA was spiked-in to a background DNA sample. Same DNA samples used in the arrays in this Series; these are technical array replicates.

Dataset Information

Improvements in sample preparation for ChIP-chip assays

Publications

Comparison of sample preparation methods for ChIP-chip assays.

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