Showing papers on "Exome sequencing published in 2006"

Sequencing genomes from single cells by polymerase cloning

Abstract: Genome sequencing currently requires DNA from pools of numerous nearly identical cells (clones), leaving the genome sequences of many difficult-to-culture microorganisms unattainable. We report a sequencing strategy that eliminates culturing of microorganisms by using real-time isothermal amplification to form polymerase clones (plones) from the DNA of single cells. Two Escherichia coli plones, analyzed by Affymetrix chip hybridization, demonstrate that plonal amplification is specific and the bias is randomly distributed. Whole-genome shotgun sequencing of Prochlorococcus MIT9312 plones showed 62% coverage of the genome from one plone at a sequencing depth of 3.5x, and 66% coverage from a second plone at a depth of 4.7x. Genomic regions not revealed in the initial round of sequencing are recovered by sequencing PCR amplicons derived from plonal DNA. The mutation rate in single-cell amplification is <2 x 10(5), better than that of current genome sequencing standards. Polymerase cloning should provide a critical tool for systematic characterization of genome diversity in the biosphere.

Methods for determining sequence variants using ultra-deep sequencing

Abstract: The claimed invention provides for new sample preparation methods enabling direct sequencing of PCR products using pyrophosphate sequencing techniques. The PCR products may be specific regions of a genome. The techniques provided in this disclosure allows for SNP (single nucleotide polymorphism) detection, classification, and assessment of individual allelic polymorphisms in one individual or a population of individuals. The results may be used for diagnostic and treatment of patients as well as assessment of viral and bacterial population identification.

Exon sequencing and high resolution haplotype analysis of ABC transporter genes implicated in drug resistance.

Abstract: Background The ATP-binding cassette (ABC) proteins are a superfamily of efflux pumps implicated as a mechanism for multidrug resistance in cytotoxic chemotherapy, immunosuppressive therapy, HIV and epilepsy. Genetic variation in P-glycoprotein, the product of the ABCB1 gene, is proposed to mediate de novo drug resistance, but associations between polymorphisms in ABCB1 and pharmacoresistance have produced conflicting results. Potential explanations for the inconsistency of results include inadequate characterization of gene structure, variation and linkage disequilibrium (LD) in ABCB1, as well as overlap in substrate specificity between ABCB1 and the various other drug transporters.Methods and results We undertook a fundamental analysis of gene structure, variation and LD in ABCB1 and four other drug transporter genes implicated in pharmacoresistance: ABCC1, ABCC2, ABCC5 and ABCB4. Manual annotation of the five genes revealed nine shorter alternative transcripts with new untranslated regions and one novel region of coding sequence, demonstrating that on-line annotations are incomplete. Sequencing of exons in 47 Caucasian individuals identified 75 novel single nucleotide polymorphisms (SNPs) previously undescribed in any public database, including 14 new coding sequence SNPs. Genotyping of 502 SNPs in 842 Caucasian individuals across the five genes revealed large blocks of high LD, and low haplotype diversity across all five genes that could be characterized by between 67 and 114 tagging SNPs, depending on the tagging criteria.Conclusion The study illustrates that publicly available data resources on genomic organization of genes and common variation can have important gaps and limitations, and establishes a comprehensive set of tagging SNPs for future association studies in pharmacoresistance. (C) 2006 Lippincott Williams & Wilkins.

High efficiency of mutation detection in type 1 stickler syndrome using a two-stage approach: vitreoretinal assessment coupled with exon sequencing for screening COL2A1.

Abstract: Stickler syndrome is a genetically heterogenous disorder that affects the ocular, skeletal, and auditory systems. To date three genes, COL2A1, COL11A1, and COL11A2, encoding the heterotypic type II/XI collagen fibrils present in vitreous and cartilage have been shown to have mutations that result in Stickler syndrome. As systemic features in this disorder are variable we have used an ophthalmic examination to differentiate those patients with a membranous vitreous phenotype associated with mutations in COL2A1, from other patients who may have mutations in other genes. Gene amplification and exon sequencing was used to screen 50 families or sporadic cases with this membranous phenotype, for mutations in COL2A1. Mutations were detected in 47 (94%) cases consisting of 166 affected and 78 unaffected individuals. We also demonstrate that the predominantly ocular form of type 1 Stickler syndrome is not confined to mutations in the alternatively spliced exon 2. Using splicing reporter constructs we demonstrate that a mutant GC donor splice site in intron 51 can be spliced normally; this contributed to the predominantly ocular phenotype in the family in which it occurred. Hum Mutat 27(7), 696–704, 2006. © 2006 Wiley-Liss, Inc.

Document Title: Mitochondrial DNA Genome Sequencing and SNP Assay Development for Increased Power of Discrimination

Abstract: This report details the work performed over a period of five years in an NIJ-funded project intended primarily to develop a practical means for accessing the large reserve of genetic variation in the ~15,000 bp mtDNA coding region, as a means for augmenting the forensic discrimination provided by sequencing the hypervariable (HV) regions of the mtDNA control region. Our approach was to focus on the relatively small number of particularly common types present in US Caucasian, African American, and Hispanic populations, where the problem of limited discrimination is concentrated in practice. We sequenced 506 entire mtDNA genomes corresponding to 56 common HV types present at 0.5% or more in the respective populations. Full genome sequencing resolved the 56 HV types into 423 haplotypes, and permitted the identification of 123 SNP sites suitable for practical assay development. Selection criteria for target SNPs included that they be present in multiple individuals, not be redundant with variation in the HV regions, and that they not occur at positions that affect either amino acid sequence or changes in structural RNA genes. Selected discriminatory SNPs were organized into panels that are specific for particular common HV types, or their near relatives. The intent was for a forensic scientist, encountering a common HV type, to be able to turn to one or two multiplex assays that represent the best chance for detecting additional discriminatory variation for the HV type in question. This was demonstrated to be a particularly effective strategy based on the observed distribution of variation in the mtDNA coding region. For Caucasian common HV types, eight multiplex allele-specific primer extension (ASPE) assays were designed, optimized, and tested. These proved to be highly suitable with regard to characteristics important for forensic mtDNA testing. Developmental validation for sensitivity, mixture detection, and degraded samples was completed for seven of the eight panels (one still in development). One multiplex has been applied in numerous case investigations, establishing a clear practical utility. Population databases were established for the completed multiplexes. Additional work on this project included generation of new control region databases, including four regional Hispanic population samples that were significantly differentiated at the haplogroup and haplotype level. Also developed, in a collaborative project, was a multiplex ASPE SNP assay for haplogroup assignment among mtDNAs of W. European origin. 2 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.

Comparative sequencing of candidate genes in complex disease

Abstract: Complex multi-factorial diseases such as cardiovascular, metabolic, neurological and respiratory disorders affect a great number of people across the world. In the post-Human Genome Sequencing era, genome wide association studies are increasingly viable alternatives to linkage approaches in locating disease genes. In addition, positional and hypothesis-driven candidate genes can be assessed for their role in susceptibility to sporadic common diseases. The efficiency and success of these approaches depend on knowledge of DNA variation and linkage disequilibrium. This thesis describes the comparative sequencing of regions across the candidate genes HTR2C, MAOA, MAOB, IDE, KIF11 and HHEX to illustrate the importance of understanding the fine scale nucleotide and LD distribution for improvements in association study design with Obesity, Depression and Alzheimers disease. In HTR2C, recombination between the commonly used nsSNP marker, Cys23Ser, and the promoter was observed (Paper I). Furthermore, nucleotide and haplotype analysis showed that gene conversion in the promoter contributed to the complexity of LD. If the functional promoter polymorphisms act in the susceptibility to serotonergic-related phenotypes, this work suggests that the unknown structure of LD across HTR2C could have been an issue in previous association studies using Cys23Ser. Support for HTR2C promoter polymorphisms in obesity was provided by the associations of promoter haplotype, TA13GCG ( P>0.0001) with high body mass index (BMI, 30 ≥ kgm-2) and promoter SNP -995G>A, (P = 0.01) with serumleptin/%body fat (Paper I). The HTR2C promoter haplotype GGCC effects were suggestive, but not significant, as having a role in depression (Paper IV). Sequence variation was scarce in the MAO regions studied. This contributes to the hypothesis that these genes are under selective pressure and that much of the variants in public databases for MAOA/B could be population specific (Paper II). Lack of MAO variation was reflected in the poor validation rate of SNPs and LD complex structure in a Swedish twin sample group. While MAOB SNPs were found to correlate with depressive state in elderly Swedish Twins, no association was observed with polymorphisms in this gene and trbc-activity (Paper II). Conversely, low trbc-activity was found to associate with MAOA SNPs and haplotypes. A potentially additive effect on the risk for depression per MAO haplotype was observed. In a larger sample, no significant associations were found with any of the MAO SNPs or haplotypes (Paper IV). However, a trend towards departures from Hardy-Weinberg Equilibrium between the genes may suggest that this region warrants further sequencing to identify potential regulatory mechanisms of MAO expression. A wealth of polymorphisms was found in re-sequencing IDE, KIF11, HHEX and conserved regions within a haplotype block associated with Alzheimers Disease. However, no significant associations between IDE and KIF11 SNPs, and Alzheimers disease were observed. These works demonstrate the advantages of re-sequencing in providing a better understanding of the various genetic factors influencing studies of polymorphisms with complex diseases. With advances in technology and throughput, sequencing will become instrumental in the location of disease genes and the identification of causative polymorphisms. List of Publications This thesis is based on the following papers, which will be referred to in the text by their Roman numerals I Shane McCarthy, Salim Mottagui-Tabar, Yumi Mizuno, Bengt Sennblad, Johan Hoffstedt, Peter Arner, Claes Wahlestedt, Björn Andersson Complex HTR2C linkage disequilibrium and promoter associations with body mass index and serum leptin Hum Genet (2005) 117: 545557 II Mårten Jansson*, Shane McCarthy*, Patrick F. Sullivan, Paul Dickman, Björn Andersson, Lars Oreland, Martin Schalling and Nancy L. Pedersen MAOA haplotypes associated with thrombocyte-MAO activity BMC Genetics 2005, 6:46 III Lars Feuk*, Shane McCarthy*, Björn Andersson, Jonathan A. Prince, and Anthony J. Brookes Mutation Screening of a Haplotype Block Around the Insulin Degrading Enzyme Gene and Association With Alzheimers Disease Am J of Med Gen Part B (Neuropsychiatric Genetics) 136B:6971 (2005) IV Shane McCarthy, Annica Dominicus, Måarten Jansson, Björn Andersson, Nancy L. Pedersen Serotonin Receptor 2C Polymorphisms and Risk for Depression in Swedish Twins

Allele-specific sequencing

Abstract: The invention relates to a novel method of allele-specific sequencing in any gene which involves the knowledge of the existing SNPs within said gene and, in particular, those SNPs that are phenotypically significant for a given condition.