58 research outputs found
Transkingdom Networks: A Systems Biology Approach to Identify Causal Members of Host-Microbiota Interactions
Improvements in sequencing technologies and reduced experimental costs have
resulted in a vast number of studies generating high-throughput data. Although
the number of methods to analyze these "omics" data has also increased,
computational complexity and lack of documentation hinder researchers from
analyzing their high-throughput data to its true potential. In this chapter we
detail our data-driven, transkingdom network (TransNet) analysis protocol to
integrate and interrogate multi-omics data. This systems biology approach has
allowed us to successfully identify important causal relationships between
different taxonomic kingdoms (e.g. mammals and microbes) using diverse types of
data
High-Throughput SuperSAGE for Digital Gene Expression Analysis of Multiple Samples Using Next Generation Sequencing
We established a protocol of the SuperSAGE technology combined with next-generation sequencing, coined “High-Throughput (HT-) SuperSAGE”. SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. In the present protocol, index (barcode) sequences are employed to discriminate tags from different samples. Such barcodes allow researchers to analyze digital tags from transcriptomes of many samples in a single sequencing run by simply pooling the libraries. Here, we demonstrated that HT-SuperSAGE provided highly sensitive, reproducible and accurate digital gene expression data. By increasing throughput for analysis in HT-SuperSAGE, various applications are foreseen and several examples are provided in the present study, including analyses of laser-microdissected cells, biological replicates and tag extraction using different anchoring enzymes
Small RNA analysis in Sindbis virus infected human HEK293 cells
In contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. Since RNAi has a well-established role in controlling infection of the alphavirus Sindbis virus (SINV) in insects, we have used this virus to investigate the role of RNAi in SINV infection of human cells
Translational Database Selection and Multiplexed Sequence Capture for Up Front Filtering of Reliable Breast Cancer Biomarker Candidates
Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples
Towards the clinical implementation of pharmacogenetics in bipolar disorder.
BackgroundBipolar disorder (BD) is a psychiatric illness defined by pathological alterations between the mood states of mania and depression, causing disability, imposing healthcare costs and elevating the risk of suicide. Although effective treatments for BD exist, variability in outcomes leads to a large number of treatment failures, typically followed by a trial and error process of medication switches that can take years. Pharmacogenetic testing (PGT), by tailoring drug choice to an individual, may personalize and expedite treatment so as to identify more rapidly medications well suited to individual BD patients.DiscussionA number of associations have been made in BD between medication response phenotypes and specific genetic markers. However, to date clinical adoption of PGT has been limited, often citing questions that must be answered before it can be widely utilized. These include: What are the requirements of supporting evidence? How large is a clinically relevant effect? What degree of specificity and sensitivity are required? Does a given marker influence decision making and have clinical utility? In many cases, the answers to these questions remain unknown, and ultimately, the question of whether PGT is valid and useful must be determined empirically. Towards this aim, we have reviewed the literature and selected drug-genotype associations with the strongest evidence for utility in BD.SummaryBased upon these findings, we propose a preliminary panel for use in PGT, and a method by which the results of a PGT panel can be integrated for clinical interpretation. Finally, we argue that based on the sufficiency of accumulated evidence, PGT implementation studies are now warranted. We propose and discuss the design for a randomized clinical trial to test the use of PGT in the treatment of BD
High-throughput 454 resequencing for allele discovery and recombination mapping in Plasmodium falciparum
<p>Abstract</p> <p>Background</p> <p>Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (<it>Plasmodium falciparum</it>) genome is crucial for understanding its evolution; however the 81% (A+T) genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites.</p> <p>Results</p> <p>We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS) sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. <it>De novo </it>assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs) and many non crossover (NCO) gene conversions throughout the genome.</p> <p>Conclusions</p> <p>By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.</p
Transposable Elements Are a Major Cause of Somatic Polymorphism in Vitis vinifera L.
Through multiple vegetative propagation cycles, clones accumulate mutations in somatic cells that are at the origin of clonal phenotypic diversity in grape. Clonal diversity provided clones such as Cabernet-Sauvignon N°470, Chardonnay N° 548 and Pinot noir N° 777 which all produce wines of superior quality. The economic impact of clonal selection is therefore very high: since approx. 95% of the grapevines produced in French nurseries originate from the French clonal selection. In this study we provide the first broad description of polymorphism in different clones of a single grapevine cultivar, Pinot noir, in the context of vegetative propagation. Genome sequencing was performed using 454 GS-FLX methodology without a priori, in order to identify and quantify for the first time molecular polymorphisms responsible for clonal variability in grapevine. New generation sequencing (NGS) was used to compare a large portion of the genome of three Pinot noir clones selected for their phenotypic differences. Reads obtained with NGS and the sequence of Pinot noir ENTAV-INRA® 115 sequenced by Velasco et al., were aligned on the PN40024 reference sequence. We then searched for molecular polymorphism between clones. Three types of polymorphism (SNPs, Indels, mobile elements) were found but insertion polymorphism generated by mobile elements of many families displayed the highest mutational event with respect to clonal variation. Mobile elements inducing insertion polymorphism in the genome of Pinot noir were identified and classified and a list is presented in this study as potential markers for the study of clonal variation. Among these, the dynamic of four mobile elements with a high polymorphism level were analyzed and insertion polymorphism was confirmed in all the Pinot clones registered in France
Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Application in Rice Marker-Assisted Breeding
Abstract
Rapid and accurate genome-wide marker detection is essential to the marker-assisted breeding and functional genomics studies. In this work, we developed an integrated software, AgroMarker Finder (AMF: http://erp.novelbio.com/AMF), for providing graphical user interface (GUI) to facilitate the recently developed restriction-site associated DNA (RAD) sequencing data analysis in rice. By application of AMF, a total of 90,743 high-quality markers (82,878 SNPs and 7,865 InDels) were detected between rice varieties JP69 and Jiaoyuan5A. The density of the identified markers is 0.2 per Kb for SNP markers, and 0.02 per Kb for InDel markers. Sequencing validation revealed that the accuracy of genome-wide marker detection by AMF is 93%. In addition, a validated subset of 82 SNPs and 31 InDels were found to be closely linked to 117 important agronomic trait genes, providing a basis for subsequent marker-assisted selection (MAS) and variety identification. Furthermore, we selected 12 markers from 31 validated InDel markers to identify seed authenticity of variety Jiaoyuanyou69, and we also identified 10 markers closely linked to the fragrant gene BADH2 to minimize linkage drag for Wuxiang075 (BADH2 donor)/Jiachang1 recombinants selection. Therefore, this software provides an efficient approach for marker identification from RAD-seq data, and it would be a valuable tool for plant MAS and variety protection
The Transcriptome of Compatible and Incompatible Interactions of Potato (Solanum tuberosum) with Phytophthora infestans Revealed by DeepSAGE Analysis
Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). Understanding the molecular basis of resistance and susceptibility to late blight is therefore highly relevant for developing resistant cultivars, either by marker-assissted selection or by transgenic approaches. Specific P. infestans races having the Avr1 effector gene trigger a hypersensitive resistance response in potato plants carrying the R1 resistance gene (incompatible interaction) and cause disease in plants lacking R1 (compatible interaction). The transcriptomes of the compatible and incompatible interaction were captured by DeepSAGE analysis of 44 biological samples comprising five genotypes, differing only by the presence or absence of the R1 transgene, three infection time points and three biological replicates. 30.859 unique 21 base pair sequence tags were obtained, one third of which did not match any known potato transcript sequence. Two third of the tags were expressed at low frequency (<10 tag counts/million). 20.470 unitags matched to approximately twelve thousand potato transcribed genes. Tag frequencies were compared between compatible and incompatible interactions over the infection time course and between compatible and incompatible genotypes. Transcriptional changes were more numerous in compatible than in incompatible interactions. In contrast to incompatible interactions, transcriptional changes in the compatible interaction were observed predominantly for multigene families encoding defense response genes and genes functional in photosynthesis and CO2 fixation. Numerous transcriptional differences were also observed between near isogenic genotypes prior to infection with P. infestans. Our DeepSAGE transcriptome analysis uncovered novel candidate genes for plant host pathogen interactions, examples of which are discussed with respect to possible function
Major prospects for exploring canine vector borne diseases and novel intervention methods using 'omic technologies
Canine vector-borne diseases (CVBDs) are of major socioeconomic importance worldwide. Although many studies have provided insights into CVBDs, there has been limited exploration of fundamental molecular aspects of most pathogens, their vectors, pathogen-host relationships and disease and drug resistance using advanced, 'omic technologies. The aim of the present article is to take a prospective view of the impact that next-generation, 'omics technologies could have, with an emphasis on describing the principles of transcriptomic/genomic sequencing as well as bioinformatic technologies and their implications in both fundamental and applied areas of CVBD research. Tackling key biological questions employing these technologies will provide a 'systems biology' context and could lead to radically new intervention and management strategies against CVBDs
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