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1.
Genome Res ; 19(9): 1527-41, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19546169

RESUMO

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.


Assuntos
Pareamento de Bases , Biologia Computacional/métodos , Variação Genética , Genoma Humano , Ligases , Análise de Sequência de DNA/métodos , África , Sequência de Bases , Genômica , Genótipo , Heterozigoto , Homozigoto , Humanos , Polimorfismo de Nucleotídeo Único , Padrões de Referência
2.
Nat Methods ; 5(7): 613-9, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18516046

RESUMO

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.


Assuntos
Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/estatística & dados numéricos , Biblioteca Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Polimorfismo de Nucleotídeo Único , Sensibilidade e Especificidade , Transdução de Sinais
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