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A human genome-wide library of local phylogeny predictions for whole-genome inference problems
Srinath Sridhar, Russell Schwartz
BMC Genomics , 2008, DOI: 10.1186/1471-2164-9-389
Abstract: In order to facilitate phylogeny-based predictions on a genomic scale, we develop a library of maximum parsimony phylogenies within local regions spanning all autosomal human chromosomes based on Haplotype Map variation data. We demonstrate the utility of this library for population genetic inferences by examining a tree statistic we call 'imperfection,' which measures the reuse of variant sites within a phylogeny. This statistic is significantly predictive of recombination rate, shows additional regional and population-specific conservation, and allows us to identify outlier genes likely to have experienced unusual amounts of variation in recent human history.Recent theoretical advances in algorithms for phylogenetic tree reconstruction have made it possible to perform large-scale inferences of local maximum parsimony phylogenies from single nucleotide polymorphism (SNP) data. As results from the imperfection statistic demonstrate, phylogeny predictions encode substantial information useful for detecting genomic features and population history. This data set should serve as a platform for many kinds of inferences one may wish to make about human population history and genetic variation.Since the first draft sequences of the human genome were completed, much of the sequencing field has turned to the problem of identifying common genomic variations and their distributions among human populations [1-3]. These variations exist predominantly in the form of single nucleotide polymorphisms (SNPs), single DNA bases that take on two common alleles in the population. While most of these variants are believed to be functionally neutral, they nonetheless encode a great deal of information about the history and structure of the population from which they are sampled, as well as the molecular evolution of the local genetic region in which each occurs. Many computational methods have been developed to infer properties of the molecular evolution or population genetics of a species
Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens
Wubin Qu, Changhong Ren, Yuan Li, Jinping Shi, Jiye Zhang, Xiaolei Wang, Xingyi Hang, Yiming Lu, Dongsheng Zhao, Chenggang Zhang
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-170
Abstract: Here we performed a reliability analysis on the Ahringer C. elegans RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3%) of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54%) bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs). The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (http://biocompute.bmi.ac.cn/CelRNAi/ webcite) was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies.Because of the potential unreliability of the Ahringer C. elegans RNAi feeding library, we strongly suggest the user examine the reliability information of the bacterial strains in the CelRNAi database before performing RNAi experiments, as well as the post-RNAi experiment analysis.Kamath and Ahringer constructed an important RNA interference (RNAi) feeding library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans in 2003 [1-3]. This RNAi feeding library has contributed largely to genome-wide functional studies of the C. elegans genes, including embryonic development [4], aging [5,6],
Identification of novel genes involved in DNA damage response by screening a genome-wide Schizosaccharomyces pombe deletion library  [cached]
Pan Xian,Lei Bingkun,Zhou Nan,Feng Biwei
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-662
Abstract: Background DNA damage response (DDR) plays pivotal roles in maintaining genome integrity and stability. An effective DDR requires the involvement of hundreds of genes that compose a complicated network. Because DDR is highly conserved in evolution, studies in lower eukaryotes can provide valuable information to elucidate the mechanism in higher organisms. Fission yeast (Schizosaccharomyces pombe) has emerged as an excellent model for DDR research in recent years. To identify novel genes involved in DDR, we screened a genome-wide S. pombe haploid deletion library against six different DNA damage reagents. The library covered 90.5% of the nonessential genes of S. pombe. Results We have identified 52 genes that were actively involved in DDR. Among the 52 genes, 20 genes were linked to DDR for the first time. Flow cytometry analysis of the repair defective mutants revealed that most of them exhibited a defect in cell cycle progression, and some caused genome instability. Microarray analysis and genetic complementation assays were carried out to characterize 6 of the novel DDR genes in more detail. Data suggested that SPBC2A9.02 and SPAC27D7.08c were required for efficient DNA replication initiation because they interacted genetically with DNA replication initiation proteins Abp1 and Abp2. In addition, deletion of sgf73+, meu29+, sec65+ or pab1+ caused improper cytokinesis and DNA re-replication, which contributed to the diploidization in the mutants. Conclusions A genome-wide screen of genes involved in DDR emphasized the key role of cell cycle control in the DDR network. Characterization of novel genes identified in the screen helps to elucidate the mechanism of the DDR network and provides valuable clues for understanding genome stability in higher eukaryotes.
Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library  [PDF]
Seiko Yoshino, Toshiro Hara, Jane S. Weng, Yuka Takahashi, Motoharu Seiki, Takeharu Sakamoto
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035590
Abstract: Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.
Evaluation of genome-wide chromatin library of Stat5 binding sites in human breast cancer
Matthew J LeBaron, Jianwu Xie, Hallgeir Rui
Molecular Cancer , 2005, DOI: 10.1186/1476-4598-4-6
Abstract: In this report, we evaluate the efficacy of a Stat5-bound chromatin library to identify valid Stat5 chromatin binding sites within the oncogenome of T-47D human breast cancer cells. A general problem with cloning of immunocaptured, transcription factor-bound chromatin fragments is contamination with non-specific chromatin. However, using an optimized strategy, five out of ten randomly selected clones could be experimentally verified to bind Stat5 both in vitro and in vivo as tested by electrophoretic mobility shift assay and chromatin immunoprecipitation, respectively. While there was no binding to fragments lacking a Stat5 consensus binding sequence, presence of a Stat5 binding sequence did not assure binding.A chromatin library coupled with experimental validation may productively identify novel in vivo Stat5 chromatin binding sites in cancer, including abnormal regulatory sites in tumor-specific neochromatin.Transcription factors function uniquely at the interface of the genome and the proteome. A significant portion of transcription factors serve not only as executors of gene transcription programs, but also as biochemical sensors of extracellular stimuli. For instance, members of the nuclear receptor family are directly activated by lipophilic extracellular ligands, and transcription factors of the Smad and Stat families are activated by phosphorylation in response to cytokine stimulation of cell surface receptors. Chromatin-bound transcription factors that act both as sensors of extracellular cues and as transcriptional effectors carry exceptional instructive value about the biological state of individual cells. Their high biological information value makes such factors particularly attractive for use as markers to predict disease activity and outcome, as well as predictive markers of disease-responsiveness to drugs.Based on related and broader rationale, the second phase of the human genome project, ENCODE (ENCyclopedia Of DNA Elements), has been initiated wi
From Human Monocytes to Genome-Wide Binding Sites - A Protocol for Small Amounts of Blood: Monocyte Isolation/ChIP-Protocol/Library Amplification/Genome Wide Computational Data Analysis  [PDF]
Sebastian Weiterer, Florian Uhle, Sabin Bhuju, Michael Jarek, Markus A. Weigand, Marek Bartkuhn
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0094164
Abstract: Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner. Conclusion: The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.
Increased Throughput by Parallelization of Library Preparation for Massive Sequencing  [PDF]
Sverker Lundin,Henrik Stranneheim,Erik Pettersson,Daniel Klevebring,Joakim Lundeberg
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0010029
Abstract: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.
Whole genome knockout library
Cathy Holding
Genome Biology , 2003, DOI: 10.1186/gb-spotlight-20031111-01
Abstract: Jacobs et al. constructed two transposons derived from Tn5 that generated alkaline phosphatase or β-galactosidase translational gene fusions when inserted into a target gene and that left a 63-bp insertion encoding an influenza hemagglutinin epitope and a hexahistidine metal affinity purification tag following cre-lox recombination. A random insertion library was generated for the MPAO1 isolate of P. aeruginosa strain PAO1 and a total of 42,240 mutant strains were archived and mapped using polymerase chain reaction. The authors observed an average success rate of 80%, of which 30,100 were unique insertions in predicted ORFs. A total of 678 candidate-essential ORFs were identified and statistical and bioinformatic analysis predicted that 300 to 400 were likely to be truly essential. Validation of the library was performed using two well characterized phenotypes: loss of surface, or 'twitching,' and auxotrophy. Of 80 'confirmed twitch' ORFs, 26 were previously known, 31 were recorded as 'mobility and attachment' genes, and 23 had no known function. Of 546 disrupted ORFs resulting in auxotrophic phenotypes, 110 were defined as 'confirmed auxotrophs,' and 58 resulted from intergenic hits."Infections with P. aeruginosa are the leading cause of death in cystic fibrosis patients, and also lead to several other clinically important infections. The development of new therapies for these infections will be challenging because of the complex biology of P. aeruginosa. The comprehensive mutant library we have constructed will allow an accelerated genetic dissection of traits such as metabolic flexibility and inherent drug resistance that make P. aeruginosa such a tenacious pathogen," the authors conclude.
The future is genome-wide
Samuel Deutsch, Alexandre Reymond
Genome Biology , 2006, DOI: 10.1186/gb-2006-7-8-324
Abstract: More than 1,700 human geneticists from 59 countries congregated in Amsterdam in May for this year's meeting of the European Society of Human Genetics, which mainly focused on the use of post-genome analysis tools to dissect the causes of and mechanisms governing complex traits. Many of the exciting studies presented were based on two technologies: array-based methods for genome-wide genotyping or technologies for high-density comparative genome hybridization (CGH). A highlight of the meeting was the keynote lecture by Nobel laureate Sydney Brenner (Salk Institute for Biological Studies, La Jolla, USA) on 'humanity's genes', which focused on the challenges we face in transforming the information from the human genome into concrete benefits for our societies.This year has seen the success of several whole-genome association studies using genotyping for single-nucleotide polymorphisms (SNPs) to identify genes responsible for some common complex phenotypes for both discrete and quantitative traits. A plenary lecture by Kari Stefansson (deCODE Genetics, Reykjavik, Iceland) highlighted the tremendous potential of this approach. Several examples were discussed in which new genes have recently been identified using a combination of linkage and association analysis approaches. One example is a locus on human chromosome 8p12 that confers susceptibility to schizophrenia. Although nucleotide variation around the NRG1 gene has been known to be associated with schizophrenia for the past 4 years, the mechanism of action of the associated SNPs, located in noncoding regions 53 to the gene, has remained unclear. Recent evidence strongly suggests that these variants might influence the level of NRG1 expression. Stefansson suggested that many SNPs involved in the etiology of complex phenotypes are likely to affect gene expression or splicing, and that these variants are under strong selective pressure. A second, more recent, example presented by Stefansson concerns the genetics of myoc
Musings on genome medicine: genome wide association studies
David G Nathan, Stuart H Orkin
Genome Medicine , 2009, DOI: 10.1186/gm3
Abstract: The development of practical approaches to DNA sequencing in the 1990s produced a remarkable scientific challenge - a proposal to establish the complete (or near complete) sequence of the human genome. Although most members of the scientific community and the media hailed the 2001 announcement of the project's initial success [1,2] as a huge intellectual and technical breakthrough, there were other voices [3]. One of us (SHO) was a member of the original US National Research Council panel that evaluated the proposal. The panel was initially highly skeptical but ended its deliberations with unbridled enthusiasm. Some leading scientists grumbled that the genome project, as it was called, was a quagmire and a money sump that had drained funds from individual investigators and provided a jumble of DNA bases the sequences of which would shed very little light on the human condition. The naysayers particularly emphasized their doubts that any medical benefit would be derived from most of the data. Indeed, when most of the human DNA sequence data had been collected, the laboratories that had accomplished the feat began to use their considerable resources to sequence the DNA of one animal species after another [4,5], with the questionable assumption that knowledge of DNA evolution would be useful and not a mere intellectual and technical exercise. Doubters began to wonder whether a large proportion of the biomedical research budget would be wasted in an effort to keep sequencing machines humming. The doubts were, in fact, quite loud in some quarters, despite the obvious fact that the project has provided investigators with ready access to all genes and facilitated positional cloning (see below).Responding to the criticism, and always ebulliently optimistic, Francis Collins, the guiding spirit of the public effort to sequence the human genome, simply changed the subject. He proposed the human HapMap project [6] to replace laborious and relatively crude restriction enzyme map
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