Showing papers on "Exome sequencing published in 2004"

Unlocking hidden genomic sequence

Abstract: Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.

Unlocking hidden genomic sequence

Abstract: Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.

Identification of two new C4 alleles by DNA sequencing and evidence for a historical recombination of serologically defined C4A and C4B alleles.

Abstract: Nucleotide polymorphisms of the C4 genes were investigated by direct sequencing of seven different homozygous typing cells from the 10IHW panels. Two novel sequences were identified within the C4d region of the C4 genes. Our sequencing analyses extend previous findings suggesting that a recombination hot spot is likely to have occurred between codon positions 1157 and 1186 within the C4d region. The classification of electrophoretically defined C4A and C4B alleles can be further subtyped by sequencing. Because the central major histocompatibility complex region that carries various copies of the C4 gene has been associated with a range of disorders; further analysis at the sequence level within the C4 locus may provide informative genetic markers for the investigation of disease-associated polymorphisms.

A new HLA-DRB1*11 allele, DRB1*1144, identified by cloning and sequencing.

Abstract: We report here the identification of a novel DRB1*11 allele, DRB1*1144, identified during sequence-based HLA-DRB1 typing. Molecular cloning and direct sequencing confirmed that the new allele is identical to DRB1*110401 at exon 2, except for a single nucleotide substitution (GTG-->GCG) changing codon 38 from Valine to Alanine.

Sequence Typing at the Speed of Light

Abstract: Pyrosequencing™ Nucleotide sequencing is an established method for gaining information relating to partial gene, whole gene, or whole genome sequence. Here we describe some of the background leading to the advent of modern nucleotide sequencing and how it has led to the development of Pyrosequencing™, a relatively new method for real-time nucleotide sequencing. In particular, we describe how this method can be used for typing bac- terial pathogens. Index Entries: Nucleotide sequencing; bacterial typing; Pyrosequencing.

The Use Of Pyrosequencing For The Analysis Of Y Chromosome Single Nucleotide Polymorphisms

Abstract: The potential value of the Y chromosome for forensic applications has been recognized for some time with the current work dedicated to Short Tandem Repeat analysis and Single Nucleotide Polymorphism (SNP) discovery. This study examined the ability of two different SNP analysis methods to determine if they could be utilized in forensic applications and ultimately be developed into an established system for Y chromosome SNP analysis. This study examined two principle SNP analysis systems: single base extension and Pyrosequencing. Pyrosequencing was determined to be superior to single base extension, due to the wealth of information provided with sequencing and the flexibility of designing primers for analysis. Using Pyrosequencing, 50 Y chromosome loci were examined and the minimum loci required for maximum diversity for the development of a Y chromosome SNP analysis system were chosen. Thirteen loci were selected based on their ability to discriminate 60 different individuals from three different racial groups into 15 different haplogroups. The Y chromosome SNP analysis system developed utilized nested PCR for the amplification of all 13 loci. Then they were sequenced as groups, ranging from one to three loci, in a single reaction. The Y chromosome SNP analysis system developed here has the potential for forensic application since it has shown to be successful in the analysis of blood, buccal swabs, semen, and saliva, works with as little as 5 pg of starting DNA material, and will amplify only male DNA in the presence of male/female mixtures in which the female portion of the sample overwhelmed the male portion 30,000 to 1. ii

Identification of the Severe Acute Respiratory Syndrome Coronavirus by Simultaneous Multigene DNA Sequencing

Abstract: The recent severe acute respiratory syndrome (SARS) outbreak resulted in calls for an accurate diagnostic test that can be used not only for routine testing but also for generating nucleotide sequences to monitor the epidemic. Although the identity of the SARS coronavirus (SARS-CoV) genome was confirmed by DNA sequencing, it is impractical to sequence the entire 29-kb SARS-CoV genome on a routine basis. Therefore, alternative assay methods such as the enzyme-linked immunosorbent assay and PCR have been pursued for routine testing, primarily to resolve probable cases. We report here a modification of standard DNA sequencing technology for accurate identification of SARS-CoV in routine testing. Instead of requiring the sequencing of the whole SARS-CoV genome, our modification enables the simultaneous sequencing of three regions of the SARS-CoV genome, the spike protein-encoding gene (35 nucleotides), gene M (43 nucleotides), and gene N (45 nucleotides), in a single electropherogram. Comparing these nucleotide sequences to DNA databank entries (National Institutes of Health) conclusively identified them as SARS-CoV sequences.

Methods and Utility of EST and Whole Genome Sequencing

Abstract: Since the Human Genome Project started in 1990 numerous large-scale genomic sequencing projects have been conducted by different consortia of laboratories. As a result, the complete genetic information of an ever growing number of higher eukaryotes is now available in the public databases, which constitute an invaluable resource for biological research. Although eukaryote genomes are very large, most of their DNA is repetitive and do not code for genes. This is particularly true for plants, where repetitive DNA can account for more than 90 % of the genome, posing a major challenge to pursue full genomic sequencing. Large-scale sequencing of cDNA (EST sequencing) is an efficient way to specifically sequence expressed genes, although no more than half of the genes can be recovered with this strategy. Gene targeted genomic sequencing approaches, like methylation filtration, overcome some of the limitations of EST sequencing and constitute an affordable alternative for plant genomic sequencing. Keywords: shotgun sequencing; genomic sequencing; expressed sequence tags; methylation filtration; physical maps