Project description:A new high-density oligonucleotide array of the human transcriptome (GG-H array) has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing, as well as detection of coding SNPs and non-coding transcripts. The GG-H array was validated using samples from multiple independent preparations of human liver and muscle, and compared with results obtained from mRNA sequencing analysis. The GG-H array is highly reproducible in estimating gene and exon abundance, and is sensitive in detecting expression changes and alternative splicing. This array has been implemented in a multi-center clinical program and has generated high quality, reproducible data. When current cost, as well as sample and time requirements for sequencing are considered in the context of a required throughput of hundreds of samples per week for a clinical trial, the array provides a high-throughput and cost effective platform for clinical genomic studies.
Project description:A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.
Project description:A new high-density oligonucleotide array of the human transcriptome (GG-H array) has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing, as well as detection of coding SNPs and non-coding transcripts. The GG-H array was validated using samples from multiple independent preparations of human liver and muscle, and compared with results obtained from mRNA sequencing analysis. The GG-H array is highly reproducible in estimating gene and exon abundance, and is sensitive in detecting expression changes and alternative splicing. This array has been implemented in a multi-center clinical program and has generated high quality, reproducible data. When current cost, as well as sample and time requirements for sequencing are considered in the context of a required throughput of hundreds of samples per week for a clinical trial, the array provides a high-throughput and cost effective platform for clinical genomic studies.
Project description:A new high-density oligonucleotide array of the human transcriptome (GG-H array) has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing, as well as detection of coding SNPs and non-coding transcripts. The GG-H array was validated using samples from multiple independent preparations of human liver and muscle, and compared with results obtained from mRNA sequencing analysis. The GG-H array is highly reproducible in estimating gene and exon abundance, and is sensitive in detecting expression changes and alternative splicing. This array has been implemented in a multi-center clinical program and has generated high quality, reproducible data. When current cost, as well as sample and time requirements for sequencing are considered in the context of a required throughput of hundreds of samples per week for a clinical trial, the array provides a high-throughput and cost effective platform for clinical genomic studies. Examination exon/gene expression of liver and muscle in quadraplicate using both the array technology and RNA-Seq
Project description:A new high-density oligonucleotide array of the human transcriptome (GG-H array) has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing, as well as detection of coding SNPs and non-coding transcripts. The GG-H array was validated using samples from multiple independent preparations of human liver and muscle, and compared with results obtained from mRNA sequencing analysis. The GG-H array is highly reproducible in estimating gene and exon abundance, and is sensitive in detecting expression changes and alternative splicing. This array has been implemented in a multi-center clinical program and has generated high quality, reproducible data. When current cost, as well as sample and time requirements for sequencing are considered in the context of a required throughput of hundreds of samples per week for a clinical trial, the array provides a high-throughput and cost effective platform for clinical genomic studies. Examination exon/gene expression of liver and muscle in quadraplicates using both the array technology and RNA-Seq
Project description:BackgroundHordeum chilense, a native South American diploid wild barley, is one of the species of the genus Hordeum with a high potential for cereal breeding purposes, given its high crossability with other members of the Triticeae tribe. Hexaploid tritordeum (×Tritordeum Ascherson et Graebner, 2n=6×=42, AABBH(ch)H(ch)) is the fertile amphiploid obtained after chromosome doubling of hybrids between Hordeum chilense and durum wheat. Approaches used in the improvement of this crop have included crosses with hexaploid wheat to promote D/H(ch) chromosome substitutions. While this approach has been successful as was the case with triticale, it has also complicated the genetic composition of the breeding materials. Until now tritordeum lines were analyzed based on molecular cytogenetic techniques and screening with a small set of DNA markers. However, the recent development of DArT markers in H. chilense offers new possibilities to screen large number of accessions more efficiently.ResultsHere, we have applied DArT markers to genotype composition in forty-six accessions of hexaploid tritordeum originating from different stages of tritordeum breeding program and to H. chilense-wheat chromosome addition lines to allow their physical mapping. Diversity analyses were conducted including dendrogram construction, principal component analysis and structure inference. Euploid and substituted tritordeums were clearly discriminated independently of the method used. However, dendrogram and Structure analyses allowed the clearest discrimination among substituted tritordeums. The physically mapped markers allowed identifying these groups as substituted tritordeums carrying the following disomic substitutions (DS): DS1D (1H(ch)), DS2D (2H(ch)), DS5D (5H(ch)), DS6D (6H(ch)) and the double substitution DS2D (2H(ch)), DS5D (5H(ch)). These results were validated using chromosome specific EST and SSR markers and GISH analysis.ConclusionIn conclusion, DArT markers have proved to be very useful to detect chromosome substitutions in the tritordeum breeding program and thus they are expected to be equally useful to detect translocations both in the tritordeum breeding program and in the transference of H. chilense genetic material in wheat breeding programs.
Project description:Transcriptome analysis is an important approach to associate genotype with phenotype. The content and dynamics of eukaryotic transcriptome are far more complex than previously anticipated. Here we integrated high-throughput RNA-seq and paired-end method to conduct an unprecedentedly deep survey of transcription profile for cultivated rice, one of the oldest domesticated crops species and has since spread worldwide to become one of the major staple foods. Analysis of reads mapping revealed 4,244 previously uncharacterized transcripts, including a mass of protein-coding genes and putative functional non-coding RNA genes. Alignment of junction reads indicated over 42% of rice multiple-exon genes produce two or more distinct splicing isoforms. It’s intriguing that we identified 1,356 putative gene fusion events, indicating the 234 fusion gene produced by trans-splicing vastly increases the complexity of rice transcriptome, together with the pervasive alternative splicing events. Digital gene expression profiling revealed most rice duplicate genes were maintained by the selection constraint on gene dosages, which would increase the genetic robustness of rice to counteract deleterious mutations
Project description:Although genomewide association studies have successfully identified associations of many common single-nucleotide polymorphisms (SNPs) with common diseases, the SNPs implicated so far account for only a small proportion of the genetic variability of tested diseases. It has been suggested that common diseases may often be caused by rare alleles missed by genomewide association studies. To identify these rare alleles we need high-throughput, high-accuracy resequencing technologies. Although array-based genotyping has allowed genomewide association studies of common SNPs in tens of thousands of samples, array-based resequencing has been limited for 2 main reasons: the lack of a fully multiplexed pipeline for high-throughput sample processing, and failure to achieve sufficient performance. We have recently solved both of these problems and created a fully multiplexed high-throughput pipeline that results in high-quality data. The pipeline consists of target amplification from genomic DNA, followed by allele enrichment to generate pools of purified variant (or nonvariant) DNA and ends with interrogation of purified DNA on resequencing arrays. We have used this pipeline to resequence approximately 5 Mb of DNA (on 3 arrays) corresponding to the exons of 1,500 genes in >473 samples; in total >2,350 Mb were sequenced. In the context of this large-scale study we obtained a false positive rate of approximately 1 in 500,000 bp and a false negative rate of approximately 10%.