Single-base resolution analysis of DNA epigenome via high-throughput sequencing

Epigenetic changes caused by DNA methylation and histone modifications play important roles in the regulation of various cellular processes and development. Recent discoveries of 5-methylcytosine (5mC) oxidation derivatives including 5-hydroxymethylcytosine (5hmC), 5-formylcytsine (5fC) and 5-carboxycytosine (5caC) in mammalian genome further expand our understanding of the epigenetic regulation. Analysis of DNA modification patterns relies increasingly on sequencing-based profiling methods. A number of different approaches have been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing (BS-seq), TAB-seq (TET-assisted bisulfite sequencing), oxBS-seq (oxidative bisulfite sequencing) and etc. These methods have been used to generate base-resolution maps of 5mC and its oxidation derivatives in genomic samples. The focus of this review will be to discuss the chemical methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.     

From biological databases to platforms for biomedical discovery

The use of high-throughput DNA sequencing and proteomic methods has led to an unprecedented increase in the amount of genomic and proteomic data. Application of computing technologies and development of computational tools to analyze and present these data has not kept pace with the accumulation of information. Here, we discuss the use of different database systems to store biological information and mention some of the key emerging computing technologies that are likely to have a key role in the future of bioinformatics.     

Optical mapping and its potential for large-scale sequencing projects.

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.     

Microsatellite markers and genetic mapping in Plasmodium falciparum

Whole-genome methods are changing the scope of biological questions that can be addressed in malaria research. In the rich context provided by Plasmodium falciparum genome sequencing, genetic mapping is a powerful tool for identifying genes involved in parasite development, invasion, transmission and drug resistance. The recent development of a high-resolution P. falciparum linkage map consisting of hundreds of microsatellite markers will facilitate an integrated genomic approach to understanding the relationship between genetic variations and biological phenotypes. Here, Michael Ferdig and Xin-zhuan Su provide an overview for applying microsatellite markers and genetic maps to gene mapping, parasite typing and studies of parasite population changes.     

MEMOSys: Bioinformatics platform for genome-scale metabolic models

Background  

Recent advances in genomic sequencing have enabled the use of genome sequencing in standard biological and biotechnological research projects. The challenge is how to integrate the large amount of data in order to gain novel biological insights. One way to leverage sequence data is to use genome-scale metabolic models. We have therefore designed and implemented a bioinformatics platform which supports the development of such metabolic models.     

Kits and their unique role in molecular biology: a brief retrospective

Since their initial development nearly 20 years ago, molecular biology kits have evolved from simple protocols and reagents for cloning of DNA to the more recent complex reagent sets that enable whole genomic sequencing. Initially met with resistance by some who felt that using them deprived researchers of the basic knowledge of how to create reagents, molecular biology kits have taken on an important role in the biological sciences. In this article we describe kit development, why kits have succeeded in molecular biology, and how they have paved the way for the more recent widespread use of core facilities.     

Genomic sequencing by ligation-mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that generates high quality, reproducible sequence ladders starting with only 1 μg of uncloned mammalian DNA per reaction. This genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures. We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method.     

Commercial high-throughput sequencing and its applications in DNA analysis

DNA sequences can be used for the analysis of genetic variation and gene function. The high-throughput sequencing techniques that have been developed over the past three years can read as many as one billion bases per run, and are far less expensive than the traditional Sanger sequencing method. Therefore, the high-throughput sequencing has been applied extensively to genomic analyses, such as screening for mutations, construction of genomic methylation maps, and the study of DNA-protein interactions. Although they have only been available for a short period, high-throughput sequencing techniques are profoundly affecting many of the life sciences, and are opening out new potential avenues of research. With the highly-developed commercial high-throughput sequencing platforms, each laboratory has the opportunity to explore this research field. Therefore, in this paper, we have focused on commercially-popular high-throughput sequencing techniques and the ways in which they have been applied over the past three years.     

Comparative genomics in vertebrate evolution and development

The vast quantities of publicly available DNA sequencing data and genome resources are enabling biologists to investigate age-old problems in biology that were not addressable previously. In this review, we discuss how comparative genomics is practiced and how the data can be used to make biological inferences with respect to vertebrate evolution and development. Examples are taken from the well-known HOX clusters, which are always a high-priority target for genomic analyses due to their inferred role in the evolution of metazoans. In addition, we briefly discuss the application of genomic approaches to problems in comparative endocrinology.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Heterogeneity and randomness of DNA methylation patterns in human embryonic stem cells

DNA methylation has been proposed to be important in many biological processes and is the subject of intense study. Traditional bisulfite genomic sequencing allows detailed high-resolution methylation pattern analysis of each molecule with haplotype information across a few hundred bases at each locus, but lacks the capacity to gather voluminous data. Although recent technological developments are aimed at assessing DNA methylation patterns in a high-throughput manner across the genome, the haplotype information cannot be accurately assembled when the sequencing reads are short or when each hybridization target only includes one or two cytosine-phosphate-guanine (CpG) sites. Whether a distinct and nonrandom DNA methylation pattern is present at a given locus is difficult to discern without the haplotype information, and the DNA methylation patterns are much less apparent because the data are often obtained only as methylation frequencies at each CpG site with some of these methods. It would facilitate the interpretation of data obtained from high-throughput bisulfite sequencing if the loci with nonrandom DNA methylation patterns could be distinguished from those that are randomly methylated. In this study, we carried out traditional genomic bisulfite sequencing using the normal diploid human embryonic stem (hES) cell lines, and utilized Hamming distance analysis to evaluate the existence of a distinct and nonrandom DNA methylation pattern at each locus studied. Our findings suggest that Hamming distance is a simple, quick, and useful tool to identify loci with nonrandom DNA methylation patterns and may be utilized to discern links between biological changes and DNA methylation patterns in the high-throughput bisulfite sequencing data sets.     

Microfluidic droplet enrichment for targeted sequencing

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications.     

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.     

Genomics: from novel genes to new therapeutics in parasitology

The advent of rapid DNA sequencing technologies is generating vast quantities of raw genomic information ranging from in-depth analysis of the expressed genes to complete sequencing of genomes at an increasing rate (bioinformatics). However, it is the functional characterisation of a specific gene product that is the key limiting factor for validation as targets for high throughput assay development. The challenge is to obtain the raw genomic information from parasites of economic importance and to effectively integrate broad technologies such as gene disruption and over-expression, DNA arrays, proteomics, antisense RNAs, with bioinformatics in a timely fashion to identify relevant biological targets. Screening of validated targets in a strategy that includes large numbers of chemistries with high diversity and predictive in vitro and in vivo assays should permit the successful identification of novel chemical entities with high specificity to the target parasite. It is proposed that this rational approach will permit the identification of new antiparasitic therapies able to surpass the current toxicological, environmental, and economic challenges of the marketplace.     

The quest for the mechanisms of life

The genomic revolution, manifested by the sequencing of the complete genome of many organisms, along with technological advances, such as DNA microarrays and developments in high-throughput analysis of proteins, metabolites, and isotopic tracer distribution patterns, challenged the conventional ways in which questions are approached in the biological sciences: (a) rather than examining a small number of genes and/or reactions at any one time;, we can now analyze gene expression and protein activity in the context of systems of interacting genes and gene products; (b) comprehensive analysis of biological systems requires the integration of all cellular fingerprints: genome sequence, maps of gene expression, protein expression, metabolic output, and in vivo enzymatic activity; and (c) collecting, managing, and analyzing comparable data from various cellular profiles requires expertise from several fields that transcend traditional discipline boundaries. While researchers in systems biology have still to address difficult challenges in both experimental and computational arenas, they possess, for the first time, the opportunity to unravel the mechanisms of life. The enormous impact of these discoveries in diverse areas, such as metabolic engineering, strain selection, drug screening and development, bioprocess development, disease prognosis and diagnosis, gene and other medical therapies, is an obvious motivation for pursuing integrated analyses of cellular systems.     

Genomic resources for flatfish research and their applications

Flatfishes are a group of teleosts of high commercial and environmental interest, whose biology is still poorly understood. The recent rapid development of different 'omic' technologies is, however, enhancing the knowledge of the complex genetic control underlying different physiological processes of flatfishes. This review describes the different functional genomic approaches and resources currently available for flatfish research and summarizes different areas where microarray-based gene expression analysis has been applied. The increase in genome sequencing data has also allowed the construction of genetic linkage maps in different flatfish species; these maps are invaluable for investigating genome organization and identifying genetic traits of commercial interest. Despite the significant progress in this field, the genomic resources currently available for flatfish are still scarce. Further intensive research should be carried out to develop larger genomic sequence databases, high-density microarrays and, more detailed, complete linkage maps, using second-generation sequencing platforms. These tools will be crucial for further expanding the knowledge of flatfish physiology, and it is predicted that they will have important implications for wild fish population management, improved fish welfare and increased productivity in aquaculture.     

Computational Prediction of Protein–Protein Interactions

Recently a number of computational approaches have been developed for the prediction of protein–protein interactions. Complete genome sequencing projects have provided the vast amount of information needed for these analyses. These methods utilize the structural, genomic, and biological context of proteins and genes in complete genomes to predict protein interaction networks and functional linkages between proteins. Given that experimental techniques remain expensive, time-consuming, and labor-intensive, these methods represent an important advance in proteomics. Some of these approaches utilize sequence data alone to predict interactions, while others combine multiple computational and experimental datasets to accurately build protein interaction maps for complete genomes. These methods represent a complementary approach to current high-throughput projects whose aim is to delineate protein interaction maps in complete genomes. We will describe a number of computational protocols for protein interaction prediction based on the structural, genomic, and biological context of proteins in complete genomes, and detail methods for protein interaction network visualization and analysis.