Project description:Next generation sequencing (NGS) technologies have impressively accelerated research in biological science during the last years by enabling the production of large volumes of sequence data to a drastically lower price per base, compared to traditional sequencing methods. The recent and ongoing developments in the field allow addressing research questions in plant-microbe biology that were not conceivable just a few years ago. The present review provides an overview of NGS technologies and their usefulness for the analysis of microorganisms that live in association with plants. Possible limitations of the different sequencing systems, in particular sources of errors and bias, are critically discussed and methods are disclosed that help to overcome these shortcomings. A focus will be on the application of NGS methods in metagenomic studies, including the analysis of microbial communities by amplicon sequencing, which can be considered as a targeted metagenomic approach. Different applications of NGS technologies are exemplified by selected research articles that address the biology of the plant associated microbiota to demonstrate the worth of the new methods.
Project description:Sequencing of the human genome has led to the definition of the genes for most of the relevant blood group systems, and the polymorphisms responsible for most of the clinically relevant blood group antigens are characterized. Molecular blood group typing is used in situations where erythrocytes are not available or where serological testing was inconclusive or not possible due to the lack of antisera. Also, molecular testing may be more cost-effective in certain situations. Molecular typing approaches are mostly based on either PCR with specific primers, DNA hybridization, or DNA sequencing. Particularly the transition of sequencing techniques from Sanger-based sequencing to next-generation sequencing (NGS) technologies has led to exciting new possibilities in blood group genotyping. We describe briefly the currently available NGS platforms and their specifications, depict the genetic background of blood group polymorphisms, and discuss applications for NGS approaches in immunohematology. As an example, we delineate a protocol for large-scale donor blood group screening established and in use at our institution. Furthermore, we discuss technical challenges and limitations as well as the prospect for future developments, including long-read sequencing technologies.
Project description:Genetic risk factors that underlie many rare and common neurological diseases remain poorly understood because of the multi-factorial and heterogeneous nature of these disorders. Although genome-wide association studies (GWAS) have successfully uncovered numerous susceptibility genes for these diseases, odds ratios associated with risk alleles are generally low and account for only a small proportion of estimated heritability. These results implicated that there are rare (present in <5% of the population) but not causative variants exist in the pathogenesis of these diseases, which usually have large effect size and cannot be captured by GWAS. With the decreasing cost of next-generation sequencing (NGS) technologies, whole-genome sequencing (WGS) and whole-exome sequencing (WES) have enabled the rapid identification of rare variants with large effect size, which made huge progress in understanding the basis of many Mendelian neurological conditions as well as complex neurological diseases. In this article, recent NGS-based studies that aimed to investigate genetic causes for neurological diseases, including Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, stroke, amyotrophic lateral sclerosis and spinocerebellar ataxias, have been reviewed. In addition, we also discuss the future directions of NGS applications in this article.
Project description:BackgroundWe have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis.ResultsThe Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics.ConclusionNG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms.
Project description:In the past decade, the development of next-generation sequencing (NGS) has paved the way for whole-genome analysis in individuals. Research on the human leukocyte antigen (HLA), an extensively studied molecule involved in immunity, has benefitted from NGS technologies. The HLA region, a 3.6-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, primarily autoimmune diseases. Recently, the HLA region has received much attention because severe adverse effects of various drugs are associated with particular HLA alleles. Owing to the complex nature of the HLA genes, classical direct sequencing methods cannot comprehensively elucidate the genomic makeup of HLA genes. Thus far, several high-throughput HLA-typing methods using NGS have been developed. In HLA research, NGS facilitates complete HLA sequencing and is expected to improve our understanding of the mechanisms through which HLA genes are modulated, including transcription, regulation of gene expression and epigenetics. Most importantly, NGS may also permit the analysis of HLA-omics. In this review, we summarize the impact of NGS on HLA research, with a focus on the potential for clinical applications.
Project description:The emergence of high-throughput next-generation sequencing technologies (e.g., 454 Life Sciences [Roche], Illumina sequencing [formerly Solexa sequencing]) has dramatically sped up whole-genome de novo sequencing and resequencing. While the low cost of these sequencing technologies provides an unparalleled opportunity for genome-wide polymorphism discovery, the analysis of the new data types and huge data volume poses formidable informatics challenges for base calling, read alignment and genome assembly, polymorphism detection, as well as data visualization. We introduce a new data integration and visualization tool EagleView to facilitate data analyses, visual validation, and hypothesis generation. EagleView can handle a large genome assembly of millions of reads. It supports a compact assembly view, multiple navigation modes, and a pinpoint view of technology-specific trace information. Moreover, EagleView supports viewing coassembly of mixed-type reads from different technologies and supports integrating genome feature annotations into genome assemblies. EagleView has been used in our own lab and by over 100 research labs worldwide for next-generation sequence analyses. The EagleView software is freely available for not-for-profit use at http://bioinformatics.bc.edu/marthlab/EagleView.
Project description:Primary immunodeficiencies (PIDs) are genetic disorders impairing host immunity, leading to life-threatening infections, autoimmunity, and/or malignancies. Genomic technologies have been critical for expediting the discovery of novel genetic defects underlying PIDs, expanding our knowledge of the complex clinical phenotypes associated with PIDs, and in shifting paradigms of PID pathogenesis. Once considered Mendelian, monogenic, and completely penetrant disorders, genomic studies have redefined PIDs as a heterogeneous group of diseases found in the global population that may arise through multigenic defects, non-germline transmission, and with variable penetrance. This review examines the uses of next-generation DNA sequencing (NGS) in the diagnosis of PIDs. While whole genome sequencing identifies variants throughout the genome, whole exome sequencing sequences only the protein-coding regions within a genome, and targeted gene panels sequence only a specific cohort of genes. The advantages and limitations of each sequencing approach are compared. The complexities of variant interpretation and variant validation remain the major challenge in wide-spread implementation of these technologies. Lastly, the roles of NGS in newborn screening and precision therapeutics for individuals with PID are also addressed.
Project description:NGPS is a method for de-novo, full-length protein sequencing in high throughput. The method is based on cleavage of the protein at semi-random sites by microwave-assisted acid hydrolysis (MAAH), enrichment of LC-MS/MS amenable peptides from the hydrolysate by solid-phase-extraction, LC-MS/MS analysis, de-novo long peptide tag sequencing of resulting peptides and assembly of peptide tags into consensus contigs.