46 research outputs found
Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays
BACKGROUND: DNA microarrays are rapidly becoming a fundamental tool in discovery-based genomic and biomedical research. However, the reliability of the microarray results is being challenged due to the existence of different technologies and non-standard methods of data analysis and interpretation. In the absence of a "gold standard"/"reference method" for the gene expression measurements, studies evaluating and comparing the performance of various microarray platforms have often yielded subjective and conflicting conclusions. To address this issue we have conducted a large scale TaqMan(® )Gene Expression Assay based real-time PCR experiment and used this data set as the reference to evaluate the performance of two representative commercial microarray platforms. RESULTS: In this study, we analyzed the gene expression profiles of three human tissues: brain, lung, liver and one universal human reference sample (UHR) using two representative commercial long-oligonucleotide microarray platforms: (1) Applied Biosystems Human Genome Survey Microarrays (based on single-color detection); (2) Agilent Whole Human Genome Oligo Microarrays (based on two-color detection). 1,375 genes represented by both microarray platforms and spanning a wide dynamic range in gene expression levels, were selected for TaqMan(® )Gene Expression Assay based real-time PCR validation. For each platform, four technical replicates were performed on the same total RNA samples according to each manufacturer's standard protocols. For Agilent arrays, comparative hybridization was performed using incorporation of Cy5 for brain/lung/liver RNA and Cy3 for UHR RNA (common reference). Using the TaqMan(® )Gene Expression Assay based real-time PCR data set as the reference set, the performance of the two microarray platforms was evaluated focusing on the following criteria: (1) Sensitivity and accuracy in detection of expression; (2) Fold change correlation with real-time PCR data in pair-wise tissues as well as in gene expression profiles determined across all tissues; (3) Sensitivity and accuracy in detection of differential expression. CONCLUSION: Our study provides one of the largest "reference" data set of gene expression measurements using TaqMan(® )Gene Expression Assay based real-time PCR technology. This data set allowed us to use an alternative gene expression technology to evaluate the performance of different microarray platforms. We conclude that microarrays are indeed invaluable discovery tools with acceptable reliability for genome-wide gene expression screening, though validation of putative changes in gene expression remains advisable. Our study also characterizes the limitations of microarrays; understanding these limitations will enable researchers to more effectively evaluate microarray results in a more cautious and appropriate manner
Polycomb-Like 3 Promotes Polycomb Repressive Complex 2 Binding to CpG Islands and Embryonic Stem Cell Self-Renewal
Polycomb repressive complex 2 (PRC2) trimethylates lysine 27 of histone H3 (H3K27me3) to regulate gene expression during diverse biological transitions in development, embryonic stem cell (ESC) differentiation, and cancer. Here, we show that Polycomb-like 3 (Pcl3) is a component of PRC2 that promotes ESC self-renewal. Using mass spectrometry, we identified Pcl3 as a Suz12 binding partner and confirmed Pcl3 interactions with core PRC2 components by co-immunoprecipitation. Knockdown of Pcl3 in ESCs increases spontaneous differentiation, yet does not affect early differentiation decisions as assessed in teratomas and embryoid bodies, indicating that Pcl3 has a specific role in regulating ESC self-renewal. Consistent with Pcl3 promoting PRC2 function, decreasing Pcl3 levels reduces H3K27me3 levels while overexpressing Pcl3 increases H3K27me3 levels. Furthermore, chromatin immunoprecipitation and sequencing (ChIP-seq) reveal that Pcl3 co-localizes with PRC2 core component, Suz12, and depletion of Pcl3 decreases Suz12 binding at over 60% of PRC2 targets. Mutation of conserved residues within the Pcl3 Tudor domain, a domain implicated in recognizing methylated histones, compromises H3K27me3 formation, suggesting that the Tudor domain of Pcl3 is essential for function. We also show that Pcl3 and its paralog, Pcl2, exist in different PRC2 complexes but bind many of the same PRC2 targets, particularly CpG islands regulated by Pcl3. Thus, Pcl3 is a component of PRC2 critical for ESC self-renewal, histone methylation, and recruitment of PRC2 to a subset of its genomic sites
Recommended from our members
The Role of Polycomp-like 3 and Ofd1 in Embryonic Stem Cell Maintenance and Differentiation
Embryonic stem cells (ESCs) hold tremendous promise for cell based therapeutics and as a biological tool. As differentiation of ESCs mimics early development, ESCs also serve as an excellent tool to study the underlying mechanisms of many developmental processes and cancer. The transition from ESC to somatic cell requires proper signaling as well as many intricate layers of gene regulation. Clarifying the interplay between these different processes is important for understanding the many transitions that happen during development as well as those that occur upon cancer metastasis., To elucidate some of the molecular mechanisms of ESC differentiation, we explored different areas of regulation within ESCs including cell signaling as well as epigenetic and transcription gene regulation. To best investigate these areas, we developed a recombinant gene technology called the Floxin system, in which we specifically and efficiently tagged genes within their endogenous loci in ESCs. The gene trap and tagged ESC lines were used to identify novel binding partners and investigate disease alleles. Through the Floxin system, we demonstrate that Ofd1, a protein required for ciliogenesis, restrains neural differentiation in embryoid bodies (EBs). We also show that Ofd1 is essential for proper Hh and Wnt signaling and that disease alleles of Ofd1 cannot rescue proper neural differentiation. Secondly, we investigate Polycomb-like 3 (Pcl3), a protein involved in histone modifications and gene repression. We found that Pcl3 incorporates into Polycomb repressive complex 2 (PRC2) and promotes complex function. Diminishment of Pcl3 decreased PRC2 binding as well as ESC self-renewal, indicating that Pcl3 enhances PRC2 targeting and ESC maintenance. Finally, we identify novel binding partners of Sall4, a transcription factor important for ESC pluripotency and reprogramming. Notably, Ofd1, Pcl3, and Sall4 are all associated with human conditions and cancer. Thus besides understanding how these proteins function in ESC maintenance and differentiation, we have gained insight into the mechanisms by which disruption of these proteins contributes to human disease
Polycomb-like 3 promotes polycomb repressive complex 2 binding to CpG islands and embryonic stem cell self-renewal.
Identifying and mitigating batch effects in whole genome sequencing data
Abstract Background Large sample sets of whole genome sequencing with deep coverage are being generated, however assembling datasets from different sources inevitably introduces batch effects. These batch effects are not well understood and can be due to changes in the sequencing protocol or bioinformatics tools used to process the data. No systematic algorithms or heuristics exist to detect and filter batch effects or remove associations impacted by batch effects in whole genome sequencing data. Results We describe key quality metrics, provide a freely available software package to compute them, and demonstrate that identification of batch effects is aided by principal components analysis of these metrics. To mitigate batch effects, we developed new site-specific filters that identified and removed variants that falsely associated with the phenotype due to batch effect. These include filtering based on: a haplotype based genotype correction, a differential genotype quality test, and removing sites with missing genotype rate greater than 30% after setting genotypes with quality scores less than 20 to missing. This method removed 96.1% of unconfirmed genome-wide significant SNP associations and 97.6% of unconfirmed genome-wide significant indel associations. We performed analyses to demonstrate that: 1) These filters impacted variants known to be disease associated as 2 out of 16 confirmed associations in an AMD candidate SNP analysis were filtered, representing a reduction in power of 12.5%, 2) In the absence of batch effects, these filters removed only a small proportion of variants across the genome (type I error rate of 3%), and 3) in an independent dataset, the method removed 90.2% of unconfirmed genome-wide SNP associations and 89.8% of unconfirmed genome-wide indel associations. Conclusions Researchers currently do not have effective tools to identify and mitigate batch effects in whole genome sequencing data. We developed and validated methods and filters to address this deficiency
Recommended from our members
Floxin, a resource for genetically engineering mouse ESCs.
We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs
The Ciliogenic Protein Oral-Facial-Digital 1 Regulates the Neuronal Differentiation of Embryonic Stem Cells
Oral-Facial-Digital 1 (OFD1) Syndrome is an X-linked developmental disorder caused by mutations in the gene Ofd1. OFD1 syndrome involves malformation of the face, oral cavity, and digits and may be characterized by cystic kidneys and mental retardation. Deletion or missense mutations in Ofd1 also result in loss of primary cilia, a microtubule-based cellular projection that mediates multiple signaling pathways. Ofd1 mutant mice display pleiotropic developmental phenotypes, including neural, skeletal, and cardiac defects. To address how loss of Ofd1 and loss of primary cilia affect early differentiation decisions, we analyzed embryoid bodies (EBs) derived from Ofd1 mutant embryonic stem (ES) cells. Ofd1 mutant EBs do not form primary cilia and display defects in Hedgehog and Wnt signaling. Additionally, we show that ES cells lacking Ofd1 display an increased capacity to differentiate into neurons. Nevertheless, neurons derived from Ofd1 mutant ES cells fail to differentiate into V3 interneurons, a cell type dependent on ciliary function and Hedgehog signaling. Thus, loss of Ofd1 affects ES cell interpretation of developmental cues and reveals that EBs model some aspects of ciliopathies, providing insights into the developmental origins of OFD1 syndrome and functions of cilia
Additional file 3: of Identifying and mitigating batch effects in whole genome sequencing data
Sample level summary statistics and annotations calculated by genotypeeval. (CSV 102Â kb
Genetic variation associated with thyroid autoimmunity shapes the systemic immune response to PD-1 checkpoint blockade
Endocrinopathies, such as thyroid autoimmunity, are common among patients treated with immune checkpoint inhibitors. Here, by using a polygenic risk score (PRS) derived from a hypothyroidism GWAS, the authors show that cancer patients with high PRS are at increased risk of atezolizumab (anti-PD-L1)-induced thyroid dysfunction, a condition associated with systemic response to PD-1 checkpoint blockade and longer overall survival