基于表观基因组学的DNA元件鉴定方法研究进展

在人类基因组测序已经完成的"后基因组"时代,对基因组序列的功能注释,尤其是各种DNA调控元件的鉴定,已成为进一步理解人类基因组复杂机制的瓶颈问题.最近,针对染色质状态图谱的大规模研究工作,揭示了各类DNA元件特征性的染色质修饰标记.这些研究结果推动了一系列基于有监督和无监督学习的DNA元件预测方法的产生,其中一些方法已经成功应用于多个基因组的DNA元件预测,并且已成为未知基因组的常规注释工具.这些预测方法因其算法特点和预测策略不同而适用于不同类型的DNA元件预测任务.大多数情况下,使用者需要联合使用多个预测方法来达到预测敏感性和特异性的平衡.尽管各类算法在DNA元件预测中都有一些成功的应用,但每一类算法都有其特有的弊端,需要使用者认真避免.本文回顾了前期和当下DNA元件预测方法的主要类型,全面分析了各类方法的优缺点,指出了下一步可以改进的方向.本综述中的分析和观点有助于读者深入理解DNA元件预测算法的主要原则,进而在相关研究中更好地应用这些方法.     

Finding regulatory sequences

DNA regulatory sequences control gene expression by forming DNA-protein complex with specific DNA binding protein. A major task of studies of gene regulation is to identify DNA regulatory sequences in genome-wide. Especially with the rapid pace of genome project, the function of DNA regulatory sequences becomes one of the focuses in functional genome era. Several approaches for screening and characterizing DNA regulatory sequences emerged one by one, from initial low-throughput methods to high-throughput strategies. Even though at present bioinformatics tools facilitate the process of screening regulatory fragments, the most reliable results will come from experimental test. This article highlights some experimental methods for the identification of regulatory sequences. A brief review of the history and procedures for selection methods are provided. Tendency as well as limitation and extension of these methods are also presented.     

Genomic strategies to identify mammalian regulatory sequences

With the continuing accomplishments of the human genome project, high-throughput strategies to identify DNA sequences that are important in mammalian gene regulation are becoming increasingly feasible. In contrast to the historic, labour-intensive, wet-laboratory methods for identifying regulatory sequences, many modern approaches are heavily focused on the computational analysis of large genomic data sets. Data from inter-species genomic sequence comparisons and genome-wide expression profiling, integrated with various computational tools, are poised to contribute to the decoding of genomic sequence and to the identification of those sequences that orchestrate gene regulation. In this review, we highlight several genomic approaches that are being used to identify regulatory sequences in mammalian genomes.     

Application of primed in situ DNA synthesis (PRINS) with telomere human commercial kit in molecular cytogenetics of Equus caballus and Sus scrofa scrofa

Recently, molecular techniques have become an indispensable tools for cytogenetic research. Especially, development of in situ techniques made possible detection at the chromosomal level, genes as well as repetitive sequences like telomeres or the DNA component of telomeres. One of these methods is primed in situ DNA synthesis (PRINS) using an oligonucleotide primer complementary to the specific DNA sequence. In this report we described application of PRINS technique with telomere human commercial kit to telomere sequences identification. This commercial kit may be use to visualization of interstitial telomeric signal in pig genome. PRINS is attractive complement to FISH for detection of DNA repetitive sequences and displays lower level of non-specific hybridization than conventional FISH.     

Chromatin structure analysis enables detection of DNA insertions into the mammalian nuclear genome

BackgroundGenetically modified organisms (GMOs) have numerous biomedical, agricultural and environmental applications. Development of accurate methods for the detection of GMOs is a prerequisite for the identification and control of authorized and unauthorized release of these engineered organisms into the environment and into the food chain. Current detection methods are unable to detect uncharacterized GMOs, since either the DNA sequence of the transgene or the amino acid sequence of the protein must be known for DNA-based or immunological-based detection, respectively.MethodsHere we describe the application of an epigenetics-based approach for the detection of mammalian GMOs via analysis of chromatin structural changes occurring in the host nucleus upon the insertion of foreign or endogenous DNA.ResultsImmunological methods combined with DNA next generation sequencing enabled direct interrogation of chromatin structure and identification of insertions of various size foreign (human or viral) DNA sequences, DNA sequences often used as genome modification tools (e.g. viral sequences, transposon elements), or endogenous DNA sequences into the nuclear genome of a model animal organism.ConclusionsThe results provide a proof-of-concept that epigenetic approaches can be used to detect the insertion of endogenous and exogenous sequences into the genome of higher organisms where the method of genetic modification, the sequence of inserted DNA, and the exact genomic insertion site(s) are unknown.General significanceMeasurement of chromatin dynamics as a sensor for detection of genomic manipulation and, more broadly, organism exposure to environmental or other factors affecting the epigenomic landscape are discussed.     

Genome annotation: which tools do we have for it?

Genome data have to be converted into knowledge to be useful to biologists. Many valuable computational tools have already been developed to help annotation of plant genome sequences, and these may be improved further, for example by identification of more gene regulatory elements. The lack of a standard computer-assisted annotation platform for eukaryotic genomes remains major bottle-neck.     

Improved repeat identification and masking in Dipterans

Repetitive sequences are a major constituent of many eukaryote genomes and play roles in gene regulation, chromosome inheritance, nuclear architecture, and genome stability. The identification of repetitive elements has traditionally relied on in-depth, manual curation and computational determination of close relatives based on DNA identity. However, the rapid divergence of repetitive sequence has made identification of repeats by DNA identity difficult even in closely related species. Hence, the presence of unidentified repeats in genome sequences affects the quality of gene annotations and annotation-dependent analyses (e.g. microarray analyses). We have developed an enhanced repeat identification pipeline using two approaches. First, the de novo repeat finding program PILER-DF was used to identify interspersed repetitive elements in several recently finished Dipteran genomes. Repeats were classified, when possible, according to their similarity to known elements described in Repbase and GenBank, and also screened against annotated genes as one means of eliminating false positives. Second, we used a new program called RepeatRunner, which integrates results from both RepeatMasker nucleotide searches and protein searches using BLASTX. Using RepeatRunner with PILER-DF predictions, we masked repeats in thirteen Dipteran genomes and conclude that combining PILER-DF and RepeatRunner greatly enhances repeat identification in both well-characterized and un-annotated genomes.     

Recently integrated human Alu repeats: finding needles in the haystack

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Computational identification of cis-acting elements affecting post-transcriptional control of gene expression in Saccharomyces cerevisiae

Understanding the regulation of gene expression requires the identification of cis -acting control elements that modulate gene function. The recent availability of complete genome sequences and profiles of mRNA expression has facilitated the development and utilization of computational methods to identify discrete regulatory elements. We have developed an oligomer counting method that identifies sequences that occur significantly more often in a group of interest relative to other genes in the genome. The use of a second parameter, which measures the frequency of oligomers within the group of interest, allows the detection of false positive signals caused by very infrequent oligomers that would otherwise appear as significant. Applying this method to gene groups that have a common expression pattern or shared function should identify oligomers that comprise cis -acting control elements. As a test of this method, we applied this approach to a set of intron-containing yeast genes, where we easily identified the known splicing signals as control elements. We have used this training set to examine how this method is affected by the length of the oligomer examined, as well as the size and composition of the gene group. These simulations allowed us to identify rules for selecting groups of genes to analyze. Finally, application of this method to nuclear genes encoding proteins targeted to the mitochondria identified a new putative cis -acting sequence in the 3'-untranslated region of this family of genes, which may play a role in mRNA localization or the regulation of mRNA stability or translation.     

Oligonucleotide fingerprint identification for microarray-based pathogen diagnostic assays

MOTIVATION: Advances in DNA microarray technology and computational methods have unlocked new opportunities to identify 'DNA fingerprints', i.e. oligonucleotide sequences that uniquely identify a specific genome. We present an integrated approach for the computational identification of DNA fingerprints for design of microarray-based pathogen diagnostic assays. We provide a quantifiable definition of a DNA fingerprint stated both from a computational as well as an experimental point of view, and the analytical proof that all in silico fingerprints satisfying the stated definition are found using our approach. RESULTS: The presented computational approach is implemented in an integrated high-performance computing (HPC) software tool for oligonucleotide fingerprint identification termed TOFI. We employed TOFI to identify in silico DNA fingerprints for several bacteria and plasmid sequences, which were then experimentally evaluated as potential probes for microarray-based diagnostic assays. Results and analysis of approximately 150 in silico DNA fingerprints for Yersinia pestis and 250 fingerprints for Francisella tularensis are presented. AVAILABILITY: The implemented algorithm is available upon request.     

Comparative genomics using fugu: a tool for the identification of conserved vertebrate cis-regulatory elements

With the imminent completion of the whole genome sequence of humans, increasing attention is being focused on the annotation of cis-regulatory elements in the human genome. Comparative genomics approaches based on evolutionary conservation have proved useful in the detection of conserved cis-regulatory elements. The pufferfish, Fugu rubripes, is an attractive vertebrate model for comparative genomics, by virtue of its compact genome and maximal phylogenetic distance from mammals. Fugu has lost a large proportion of nonessential DNA, and retained single orthologs for many duplicate genes that arose in the fish lineage. Non-coding sequences conserved between fugu and mammals have been shown to be functional cis-regulatory elements. Thus, fugu is a model fish genome of choice for discovering evolutionarily conserved regulatory elements in the human genome. Such evolutionarily conserved elements are likely to be shared by all vertebrates, and related to regulatory interactions fundamental to all vertebrates. The functions of these conserved vertebrate elements can be rapidly assayed in mammalian cell lines or in transgenic systems such as zebrafish/medaka and Xenopus, followed by validation of crucial elements in transgenic rodents.