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Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription [RNA-Seq]

ABSTRACT: The application of massively-parallel sequencing (MPS) technology to understanding how the genome is regulated is of major importance, as we now appreciate sequence variants associated with common heritable diseases to be enriched at regulatory elements in the genome. There is, however, an enormous challenge inherent to these studies, which are much more varied as assays and in terms of analytical approaches than the approaches used to define the sequence variants themselves. For example, there areis a plethora of assays that test 5-methylcytosine genome-wide, numerous different analytical approaches to defineing peaks from chromatin immunoprecipitation sequencing (ChIP-seq), and increasingly diverse aspects of genomic regulation being studied. The extremely large data sets generated do not lend themselves to distribution without some degree of pre-processing, and the hardware requirements for running these analyses are often substantial. We have highlighted these concerns previously, but the central concern for our field is that genomics research is especially prone to poor reproducibility, and is a prime candidate for implementation of measures that could help to address this general problem.To illustrate the practical issues involved in genomics reproducibility, we sought to analyze our and three previoulsy published studies of the histone demethylase KDM5B using the same analytical approaches.

ORGANISM(S): Mus musculus

PROVIDER: GSE63285 | GEO | 2020/03/21

REPOSITORIES: GEO

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Project description:The accurate mapping of recurring DNA copy number aberrations (CNAs), a hallmark feature of the cancer genome, has facilitated the discovery of tumor suppressor genes and oncogenes. Microarray-based assays designed to detect these chromosomal copy number alterations on a genome-wide and high-resolution scale have emerged as a cornerstone technology in the genomic era. The diversity of commercially-available platforms prompted a systematic comparison of five copy number profiling assays for their ability to detect 2-fold copy number gain and loss (4n or 1n, respectively) as well as focal high-amplitude CNAs. Here, using a collection of established human melanoma cell lines, we defined the reproducibility, absolute signals, signal:noise, false-positive and false-negative rates for each of the five assays against ground-truth defined by Spectral Karyotyping (SKY), in addition to comparing the concordance of CNAs detection by two high-resolution Agilent and Affymetrix microarray platforms. Our analyses concluded that the Agilentâs 60mer oligo-microarray with probe design optimized for genomic hybridization offers the highest sensitivity and specificity [area under Receiver Operator Characteristic (ROC) curve >0.99] while Affymetrixâs SNP microarray appears to offer better detection of CNAs in gene-poor region. Availability of these comparison results should guide study design decisions and facilitate further computational development. Keywords: comparative genomic hybridization We generated copy number profiles of a defined set of tumor cell lines on five oligo-microarray-based assays of three platforms (Agilent, Affymetrix and NimbleGen) and determined the reproducibility, signal and noise as well as sensitivity and specificity of each in detecting two-fold signals based on SKY-defined aberrations as ground-truth for comparison. In addition, high-density microarray assays from Agilent and Affymetrix platforms were further compared for definition of CNAs in an independent dataset using published analytical approaches. All arrays were run from the same DNA harvest from respective cell lines. Each array was run according to the protocol of the manufacturer.

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Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription [RNA-Seq]

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