detection-rare-genomic-variants-from-pooled-sequencing-using

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Washington University School of Medicine

Ohno [Ohno, S. (1970) in Evolution by Gene Duplication, Springer, New York] proposed that gene duplication with subsequent divergence of paralogs could be a major force in the evolution of new gene functions. In practice the functional differences between closely related homologues produced by duplications can be subtle and difficult to separate experimentally. Here we show that DNA microarrays can distinguish the functions of two closely related homologues from the yeast Saccharomyces cerevisiae, Yap1p and Yap2p. Although Yap1p and Yap2p are both bZIP transcription factors involved in multiple stress responses and are 88% identical in their DNA binding domains, our work shows that these proteins activate nonoverlapping sets of genes. Yap1p controls a set of genes involved in detoxifying the effects of reactive oxygen species, whereas Yap2p controls a set of genes over represented for the function of stabilizing proteins. In addition we show that the binding sites in the promoters of the Yap1p-dependent genes differ from the sites in the promoters of Yap2p-dependent genes and we validate experimentally that these differences are important for regulation by Yap1p. We conclude that while Yap1p and Yap2p may have some overlapping functions they are clearly not redundant and, more generally, that DNA microarray analysis will be an important tool for distinguishing the functions of the large numbers of highly conserved genes found in all eukaryotic genomes.

Discrimination between paralogs using microarray analysis: application to the Yap1p and Yap2p transcriptional networks.

Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two single- nucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.

Digital genotyping and haplotyping with polymerase colonies.

We describe a strategy to analyze the impact of single nucleotide mutations on protein function. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools and polyacrylamide gel immobilized PCR. A system was constructed in which the yeast PGK1 gene was expressed from a plasmid-borne copy of the gene in a PGK1 deletion strain of Saccharomyces cerevisiae. Using this system, we demonstrated that the enrichment or depletion of PGK1 point mutants from a mixed culture was consistent with the expected results based on the isolated growth rates of the mutants. Enrichment or depletion of individual point mutants was shown to result from increases or decreases, respectively, in the specific activities of the encoded proteins. Further, we demonstrate the ability to analyze the functional effect of many individual point mutations in parallel. By functional complementation of yeast deletions with human homologs, our technique could be readily applied to the functional analysis of single nucleotide polymorphisms in human genes of medical interest.

Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies.

Advanced sequencing technologies: methods and goals.

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

Accurate multiplex polony sequencing of an evolved bacterial genome.

We report a method for multilocus long-range haplotyping on human chromosome molecules in vitro based on the DNA polymerase colony (polony) technology. By immobilizing thousands of intact chromosome molecules within a polyacrylamide gel on a microscope slide and performing multiple amplifications from single molecules, we determined long-range haplotypes spanning a 153-Mb region of human chromosome 7 and found evidence of rare mitotic recombination events in human lymphocytes. Furthermore, the parallel nature of DNA polony technology allows efficient haplotyping on pooled DNAs from a population on one slide, with a throughput three orders of magnitudes higher than current molecular haplotyping methods. Linkage disequilibrium statistics established by our pooled DNA haplotyping method are more accurate than statistically inferred haplotypes. This haplotyping method is well suited for candidate gene-based association studies as well as for investigating the pattern of recombination in mammalian cells.

Long-range polony haplotyping of individual human chromosome molecules.

We report a targeted, cost-effective method to quantify rare single-nucleotide polymorphisms from pooled human genomic DNA using second-generation sequencing. We pooled DNA from 1,111 individuals and targeted four genes to identify rare germline variants. Our base-calling algorithm, SNPSeeker, derived from large deviation theory, detected single-nucleotide polymorphisms present at frequencies below the raw error rate of the sequencing platform.

Quantification of rare allelic variants from pooled genomic DNA.

Sporadic heart failure is thought to have a genetic component, but the contributing genetic events are poorly defined. Here, we used ultra-high-throughput resequencing of pooled DNAs to identify SNPs in 4 biologically relevant cardiac signaling genes, and then examined the association between allelic variants and incidence of sporadic heart failure in 2 large Caucasian populations. Resequencing of DNA pools, each containing DNA from approximately 100 individuals, was rapid, accurate, and highly sensitive for identifying common and rare SNPs; it also had striking advantages in time and cost efficiencies over individual resequencing using conventional Sanger methods. In 2,606 individuals examined, we identified a total of 129 separate SNPs in the 4 cardiac signaling genes, including 23 nonsynonymous SNPs that we believe to be novel. Comparison of allele frequencies between 625 Caucasian nonaffected controls and 1,117 Caucasian individuals with systolic heart failure revealed 12 SNPs in the cardiovascular heat shock protein gene HSPB7 with greater proportional representation in the systolic heart failure group; all 12 SNPs were confirmed in an independent replication study. These SNPs were found to be in tight linkage disequilibrium, likely reflecting a single genetic event, but none altered amino acid sequence. These results establish the power and applicability of pooled resequencing for comparative SNP association analysis of target subgenomes in large populations and identify an association between multiple HSPB7 polymorphisms and heart failure.

Cardiac signaling genes exhibit unexpected sequence diversity in sporadic cardiomyopathy, revealing HSPB7 polymorphisms associated with disease.

&#x6458;&#x8981;&#xFF1A; &#x80CC;&#x666F;&#xFF1A; &#x63D2;&#x5165;&#x5143;&#x7D20; IS6110 &#x662F;&#x4EBA;&#x7C7B;&#x7ED3;&#x6838;&#x75C5;&#x7684;&#x75C5;&#x539F;&#x4F53;&#x7ED3;&#x6838;&#x5206;&#x679D;&#x6746;&#x83CC;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x53D8;&#x5F02;&#x7684;&#x4E3B;&#x8981;&#x6765;&#x6E90;&#x4E4B;&#x4E00;&#x3002;&#x867D;&#x7136; IS6110 &#x5DF2;&#x88AB;&#x5E7F;&#x6CDB;&#x7528;&#x4F5C;&#x6D41;&#x884C;&#x75C5;&#x5B66;&#x7684;&#x6807;&#x8BB0;&#xFF0C;&#x786E;&#x5B9A;&#x7CBE;&#x786E;&#x7684;&#x67D3;&#x8272;&#x4F53;&#x63D2;&#x5165;&#x7684;&#x5730;&#x70B9;&#x53D7;&#x5230;&#x9650;&#x5236;&#x7684;&#x6280;&#x672F;&#x6311;&#x6218;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x4ECB;&#x7ECD;&#x4E86; ISseq&#xFF0C;&#x4E00;&#x79CD;&#x65B0;&#x7684;&#x65B9;&#x6CD5;&#x76F8;&#x7ED3;&#x5408;&#x4F7F;&#x7528;&#x7167;&#x6392;&#x80F6;&#x7247;&#x6280;&#x672F;&#x4E0E;&#x9AD8;&#x6548;&#x7EC4;&#x5408;&#x793A;&#x4F8B;&#x591A;&#x8DEF;&#x590D;&#x7528;&#x6765;&#x540C;&#x65F6;&#x63A2;&#x6D4B; 519 &#x7684;&#x4E34;&#x5E8A;&#x5206;&#x79BB;&#x7684;&#x9AD8;&#x901A;&#x91CF;&#x6D4B;&#x5E8F;&#x786E;&#x5B9A;&#x5355;&#x4E2A;&#x5B9E;&#x9A8C;&#x4E2D;&#x7684;&#x5143;&#x7D20;&#x7684;&#x51E0;&#x4E4E;&#x6240;&#x6709;&#x4FA7;&#x7FFC;&#x5730;&#x533A;&#x3002;&#x7ED3;&#x679C;&#xFF1A; &#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x4E0D;&#x540C;&#x5206;&#x79BB;&#x682A; 6,976 IS6110 &#x4FA7;&#x7FFC;&#x5730;&#x533A;&#x603B;&#x6570;&#x3002;&#x5F53;&#x9A8C;&#x8BC1;&#x4F7F;&#x7528;&#x53C2;&#x8003;&#x83CC;&#x682A;&#xFF0C;&#x8BE5;&#x65B9;&#x6CD5;&#x5DF2; 100%&#x7279;&#x5F02;&#x6027;&#x548C; 98%&#x7684;&#x79EF;&#x6781;&#x9884;&#x6D4B;&#x503C;&#x3002;&#x63D2;&#x5165;&#x5230;&#x7F16;&#x7801;&#x548C;&#x975E;&#x7F16;&#x7801;&#x6620;&#x5C04;&#x533A;&#x57DF;&#xFF0C;&#x5E76;&#x5728;&#x67D0;&#x4E9B;&#x60C5;&#x51B5;&#x4E0B;&#x4E2D;&#x65AD;&#x8BA4;&#x4E3A;&#x662F;&#x91CD;&#x8981;&#x7684;&#x6BD2;&#x529B;&#x6216;&#x4F53;&#x5916;&#x751F;&#x957F;&#x7684;&#x57FA;&#x56E0;&#x3002;&#x682A;&#x88AB;&#x5206;&#x4E3A;&#x5BB6;&#x5EAD;&#x4F7F;&#x7528;&#x63D2;&#x5165;&#x7684;&#x7AD9;&#x70B9;&#xFF0C;&#x800C;&#x4E14;&#x9AD8;&#x534F;&#x8BAE;&#x4E0E;&#x4EE5;&#x524D;&#x7684;&#x7814;&#x7A76;&#x89C2;&#x5BDF;&#x7ED3;&#x8BBA;&#xFF1A; &#x8FD9;&#x79CD;&#x9AD8;&#x541E;&#x5410;&#x91CF;&#x662F; seq &#x65B9;&#x6CD5;&#xFF0C;&#x8FD8;&#x53EF;&#x4EE5;&#x7528;&#x4E8E;&#x6620;&#x5C04;&#x5176;&#x4ED6;&#x6709;&#x673A;&#x4F53;&#x4E2D;&#x7684;&#x63D2;&#x5165;&#x5185;&#x5BB9;&#xFF0C;&#x6269;&#x5C55;&#x4E86;&#x5148;&#x524D;&#x8C03;&#x67E5;&#x7684;&#x4F53;&#x5185;&#x4E2D;&#x65AD;&#x57FA;&#x56E0;&#x4F4D;&#x70B9;&#x548C;&#x63A2;&#x6D4B; M.&#x80BA;&#x7ED3;&#x6838;&#x83CC;&#x682A;&#x5728;&#x4E2D;&#x65AD;&#x7684;&#x540E;&#x679C;&#x63D0;&#x4F9B;&#x4E86;&#x4E00;&#x4E2A;&#x57FA;&#x51C6;&#x3002;

IS-seq&#xFF1A; &#x65B0;&#x578B;&#x9AD8;&#x541E;&#x5410;&#x91CF;&#x7684;&#x8C03;&#x67E5;&#x4F53;&#x5185; IS6110 &#x6362;&#x4F4D;&#x591A;&#x4E2A;&#x5206;&#x679D;&#x6746;&#x83CC;&#x80BA;&#x7ED3;&#x6838;&#x57FA;&#x56E0;&#x7EC4;&#x4E2D;&#x3002;

ABSTRACT: BACKGROUND: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present ISseq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. RESULTS: We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100 % specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed CONCLUSIONS: This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.

IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes.

R&eacute;sum&eacute; : Contexte : l&#39;&eacute;l&eacute;ment d&#39;insertion IS6110 est l&#39;une des principales sources de variabilit&eacute; g&eacute;nomique de Mycobacterium tuberculosis, l&#39;agent &eacute;tiologique de la tuberculose humaine. Bien que IS6110 a &eacute;t&eacute; employ&eacute;e intensivement comme un marqueur &eacute;pid&eacute;miologique, l&#39;identification des sites pr&eacute;cis insertion chromosomique a &eacute;t&eacute; limit&eacute;e par les d&eacute;fis techniques. Nous pr&eacute;sentons ici, ISseq, une nouvelle m&eacute;thode qui combine le s&eacute;quen&ccedil;age haut d&eacute;bit utilisant la technologie d&#39;Illumina avec multiplexage efficace &eacute;chantillon combinatoire de sonder simultan&eacute;ment 519 isolats cliniques, identifier presque toutes les r&eacute;gions adjacentes de l&#39;&eacute;l&eacute;ment dans une exp&eacute;rience simple. R&Eacute;SULTATS : Nous avons identifi&eacute; un total de 6 976 IS6110 r&eacute;gions flanquantes sur les diff&eacute;rents isolats. Lors de la validation &agrave; l&#39;aide de souches de r&eacute;f&eacute;rence, la m&eacute;thode a 100 % sp&eacute;cificit&eacute; et 98 % valeur pr&eacute;dictive positive. Les insertions mapp&eacute;es &agrave; la fois codage et de non-codage r&eacute;gions et dans certains cas interrompu g&egrave;nes consid&eacute;r&eacute;s comme essentiel pour la virulence ou la croissance in vitro. Les souches ont &eacute;t&eacute; class&eacute;s en familles en utilisant des sites d&#39;insertion, et bon accord avec des &eacute;tudes ant&eacute;rieures a &eacute;t&eacute; observ&eacute; des CONCLUSIONS : cette m&eacute;thode de IS-seq haut d&eacute;bit, qui peut &eacute;galement servir pour mapper les insertions dans d&#39;autres organismes, s&#39;&eacute;tend des sondages pr&eacute;c&eacute;dents de loci interrompu in vivo et constitue une base pour sonder les cons&eacute;quences des perturbations dans les souches de M. tuberculosis.

IS-seq : une enqu&ecirc;te de roman &agrave; haut d&eacute;bit de la transposition de IS6110 in vivo dans les g&eacute;nomes de la tuberculose Mycobacterium multiples.

ABSTRACT: BACKGROUND: das Einf&uuml;gen Element IS6110 ist eine der Hauptquellen der genomischer Variabilit&auml;t in der &auml;tiologische Agent der menschlichen Tuberkulose, Mycobacterium Tuberculosis. Obwohl IS6110 ausgiebig als epidemiologische Marker verwendet wurde, wurde die Bestimmung der pr&auml;zise chromosomalen Einf&uuml;gung-Standorte durch technische Herausforderungen begrenzt. Hier pr&auml;sentieren wir ISseq, eine neuartige Methode, Hochdurchsatz-Sequenzierung mit Illumina-Technologie mit effizienten kombinatorische Beispiel Multiplexen, gleichzeitig 519 klinische Isolaten, Sonde kombiniert, fast alle flankierenden Ermittlung des-Elements in einem einzigen Experiment. Ergebnisse: Wir haben insgesamt 6.976 IS6110 flankierenden Regionen auf die verschiedenen Isolaten identifiziert. Wenn mit Referenzst&auml;mme validiert, hatte die Methode 100 % Spezifit&auml;t und 98 % positiven Vorhersagewert. Einf&uuml;gungen zugeordnete Codierung sowohl nicht-kodierenden Regionen, und in einigen F&auml;llen unterbrochen Gene notwendig f&uuml;r Virulenz bzw. f&uuml;r in-vitro-Wachstum betrachtet. Belastungen in Familien mit Einf&uuml;gung Sites eingestuft wurden und hohe &Uuml;bereinstimmung mit fr&uuml;heren Studien wurde beobachtet, Schlussfolgerungen: Diese Hochdurchsatz-IS-Seq-Methode, die auch verwendet werden kann, ordnen Sie die Einf&uuml;gungen in anderen Organismen fr&uuml;here Erhebungen in-vivo unterbrochenen Loci erweitert und bildet die Grundlage f&uuml;r die Folgen von St&ouml;rungen in M. Tuberkulose-St&auml;mme zu sondieren.

IS-Seq: eine neuartige high-Throughput-Umfrage der in-vivo IS6110-Umsetzung in mehreren Mycobacterium-Tuberkulose-Genome.

ABSTRACT: BACKGROUND: l&#39;elemento di inserimento IS6110 &egrave; una delle principali fonti di variabilit&agrave; genomica in Mycobacterium tuberculosis, agente eziologico della tubercolosi umana. Sebbene IS6110 &egrave; stato usato estesamente come un marcatore epidemiologico, l&#39;identificazione dei siti di inserzione cromosomica preciso ha stato limitato da sfide tecniche. Presentiamo qui, ISseq, un nuovo metodo che combina high-throughput sequencing utilizzando la tecnologia Illumina con efficiente campione combinatorio multiplexing per sondare contemporaneamente 519 isolati clinici, identificare quasi tutte le regioni fiancheggianti dell&#39;elemento in un singolo esperimento. RISULTATI: Abbiamo identificato un totale di 6.976 IS6110 regioni fiancheggianti sui diversi isolati. Quando convalidate utilizzando ceppi di riferimento, il metodo aveva valore predittivo 100% specificit&agrave; e 98% positivo. Le inserzioni mappate alla codifica sia non codificante regioni e in alcuni casi, interrotta geni essenziali per la virulenza o la crescita in vitro. Ceppi sono stati classificati in famiglie utilizzando siti di inserimento, e alta accordo con studi precedenti &egrave; stato osservato conclusioni: questo metodo &egrave;-seq ad alta velocit&agrave;, che pu&ograve; anche essere utilizzato per mappare gli inserimenti in altri organismi, estende le precedenti indagini in vivo loci interrotto e fornisce una base per sondare le conseguenze delle interruzioni nei ceppi di M. tuberculosis.

IS-seq: un&#39;indagine di romanzo elevato throughput di in vivo IS6110 trasposizione in multiple genomes di Mycobacterium tuberculosis.

&#x8981;&#x65E8;&#xFF1A; &#x80CC;&#x666F;&#xFF1A; &#x633F;&#x5165;&#x8981;&#x7D20; IS6110 &#x7D50;&#x6838;&#x83CC;&#x3001;&#x75C5;&#x539F;&#x4F53;&#x306E;&#x3072;&#x3068;&#x7D50;&#x6838;&#x306B;&#x304A;&#x3051;&#x308B;&#x30B2;&#x30CE;&#x30E0;&#x5909;&#x52D5;&#x306E;&#x4E3B;&#x306A;&#x60C5;&#x5831;&#x6E90;&#x306E;&#x4E00;&#x3064;&#x3067;&#x3059;&#x3002;IS6110 &#x75AB;&#x5B66;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068;&#x3057;&#x3066;&#x5E83;&#x304F;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x304C;&#x3001;&#x6B63;&#x78BA;&#x306A;&#x67D3;&#x8272;&#x4F53;&#x633F;&#x5165;&#x90E8;&#x4F4D;&#x306E;&#x540C;&#x5B9A;&#x6280;&#x8853;&#x4E0A;&#x306E;&#x8AB2;&#x984C;&#x306B;&#x3088;&#x3063;&#x3066;&#x5236;&#x9650;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x5358;&#x4E00;&#x306E;&#x5B9F;&#x9A13;&#x5185;&#x306E;&#x8981;&#x7D20;&#x306E;&#x4E26;&#x3076;&#x307B;&#x307C;&#x3059;&#x3079;&#x3066;&#x306E;&#x9818;&#x57DF;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B; ISseq&#x3001;&#x52B9;&#x7387;&#x7684;&#x306A;&#x7D44;&#x5408;&#x305B;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x306E;&#x591A;&#x91CD;&#x5316;&#x30A4;&#x30EB;&#x30DF;&#x30CA;&#x6280;&#x8853; 519 &#x306E;&#x81E8;&#x5E8A;&#x5206;&#x96E2;&#x682A;&#x3092;&#x540C;&#x6642;&#x306B;&#x30D7;&#x30ED;&#x30FC;&#x30D6;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x305F;&#x624B;&#x6CD5;&#x3092;&#x63D0;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D50;&#x679C;: 6,976 IS6110 &#x7570;&#x306A;&#x308B;&#x5206;&#x96E2;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x9818;&#x57DF;&#x306E;&#x5408;&#x8A08;&#x3092;&#x8B58;&#x5225;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x30EA;&#x30D5;&#x30A1;&#x30EC;&#x30F3;&#x30B9;&#x682A;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x691C;&#x8A3C;&#x3059;&#x308B;&#x3068;&#x304D;&#x3001;&#x30E1;&#x30BD;&#x30C3;&#x30C9; 100 &#xFF05; &#x7279;&#x7570;&#x6027;&#x3068; 98 &#xFF05; &#x306E;&#x80AF;&#x5B9A;&#x7684;&#x306A;&#x4E88;&#x6E2C;&#x5024;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x305F;&#x3002;&#x7B26;&#x53F7;&#x5316;&#x3068;&#x975E;&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x306E;&#x4E21;&#x65B9;&#x306B;&#x30DE;&#x30C3;&#x30D7;&#x306E;&#x633F;&#x5165;&#x9818;&#x57DF;&#x3001;&#x304A;&#x3088;&#x3073;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30B1;&#x30FC;&#x30B9;&#x3067;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x75C5;&#x539F;&#x6027;&#x3084; in vitro &#x306E;&#x6210;&#x9577;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3068;&#x8003;&#x3048;&#x304C;&#x4E2D;&#x65AD;&#x3057;&#x307E;&#x3059;&#x3002;&#x7CFB;&#x7D71;&#x306F;&#x3001;&#x633F;&#x5165;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x5BB6;&#x65CF;&#x306B;&#x5206;&#x985E;&#x3055;&#x308C;&#x305F;&#x3001;&#x9AD8;&#x3044;&#x5951;&#x7D04;&#x306F;&#x4EE5;&#x524D;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x7D50;&#x8AD6;&#x304C;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x305F;: &#x4ED6;&#x306E;&#x751F;&#x7269;&#x306E;&#x633F;&#x5165;&#x3092;&#x30DE;&#x30C3;&#x30D7;&#x3059;&#x308B;&#x3082;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x307E;&#x3059;&#x3001;&#x3053;&#x306E;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8; IS seq &#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x306F;&#x3001;in vivo &#x3067;&#x4E2D;&#x65AD;&#x3055;&#x308C;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x5EA7;&#x306E;&#x524D;&#x306E;&#x8ABF;&#x67FB;&#x3092;&#x62E1;&#x5F35;&#x3057;&#x3001;M. &#x7D50;&#x6838;&#x83CC;&#x682A;&#x306E;&#x6DF7;&#x4E71;&#x306E;&#x7D50;&#x679C;&#x306E;&#x30D7;&#x30ED;&#x30FC;&#x30D3;&#x30F3;&#x30B0;&#x306E;&#x305F;&#x3081;&#x306E;&#x30D9;&#x30FC;&#x30B9;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

IS-seq: &#x8907;&#x6570;&#x306E;&#x30DE;&#x30A4;&#x30B3;&#x30D0;&#x30AF;&#x30C6;&#x30EA;&#x30A6;&#x30E0;&#x306E;&#x7D50;&#x6838;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x3067;&#x306E; in vivo &#x306E; IS6110 &#x8EE2;&#x4F4D;&#x306E;&#x65B0;&#x898F;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8;&#x8ABF;&#x67FB;&#x3002;

&#xAC1C;&#xC694;: &#xBC30;&#xACBD;: &#xC0BD;&#xC785; &#xC694;&#xC18C; IS6110 &#xACB0;&#xD575;&#xADE0;, &#xC778;&#xAC04;&#xC758; &#xACB0;&#xD575; etiological &#xC5D0;&#xC774;&#xC804;&#xD2B8; &#xAC8C;&#xB188; &#xB2E4;&#xC591;&#xC131;&#xC758; &#xC8FC;&#xC694; &#xCD9C;&#xCC98; &#xC911; &#xD558;&#xB098;&#xC785;&#xB2C8;&#xB2E4;. &#xBE44;&#xB85D; IS6110 &#xC5ED;&#xD559; &#xD45C;&#xC2DD;&#xC73C;&#xB85C; &#xAD11;&#xBC94;&#xC704; &#xD558; &#xAC8C; &#xC0AC;&#xC6A9; &#xB41C; &#xC815;&#xD655;&#xD55C; &#xC5FC;&#xC0C9;&#xCCB4; &#xC0BD;&#xC785; &#xC0AC;&#xC774;&#xD2B8;&#xC758; &#xC2DD;&#xBCC4; &#xAE30;&#xC220; &#xACFC;&#xC81C;&#xC5D0; &#xC758;&#xD574; &#xC81C;&#xD55C; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC120;&#xBB3C;&#xC774; ISseq, &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; &#xC2DC;&#xD000;&#xC2F1; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xC870;&#xD569; &#xC0D8;&#xD50C; &#xB2E4;&#xC911;&#xD654;&#xC640; Illumina &#xAE30;&#xC220;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB3D9;&#xC2DC;&#xC5D0; 519 &#xC784;&#xC0C1; &#xACA9;&#xB9AC; &#xD504;&#xB85C;&#xBE0C;&#xB97C; &#xACB0;&#xD569; &#xD558; &#xC5EC; &#xC0C8;&#xB85C;&#xC6B4; &#xBC29;&#xBC95; &#xB2E8;&#xC77C; &#xC2E4;&#xD5D8;&#xC5D0;&#xC11C; &#xC694;&#xC18C;&#xC758; flanking &#xAC70;&#xC758; &#xBAA8;&#xB4E0; &#xC601;&#xC5ED;&#xC744; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;. &#xACB0;&#xACFC;: &#xCD1D; &#xB2E4;&#xB978; &#xACA9;&#xB9AC;&#xC5D0; 6,976 IS6110 flanking &#xC601;&#xC5ED; &#xC2DD;&#xBCC4;. &#xCC38;&#xC870; &#xC885;&#xC790;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xAC80;&#xC0AC; &#xD558;&#xB294; &#xACBD;&#xC6B0; &#xBA54;&#xC11C;&#xB4DC;&#xB97C; 100% &#xD2B9;&#xC774;&#xC131; &#xBC0F; 98% &#xAE0D;&#xC815;&#xC801;&#xC778; &#xC608;&#xCE21; &#xAC12;&#xC744; &#xD588;&#xB2E4;. &#xC0BD;&#xC785; &#xBAA8;&#xB450; &#xCF54;&#xB529;&#xACFC; &#xBE44; &#xCF54;&#xB529;&#xC5D0; &#xB9E4;&#xD551;&#xB41C; &#xC601;&#xC5ED;, &#xC5B4;&#xB5A4; &#xACBD;&#xC6B0;&#xC5D0; &#xC720;&#xC804;&#xC790; &#xB3C5;&#xC131; &#xB610;&#xB294; &#xCCB4; &#xC678; &#xC131;&#xC7A5;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778; &#xAC83;&#xC73C;&#xB85C; &#xC0DD;&#xAC01;&#xC744; &#xC911;&#xB2E8; &#xD558; &#xACE0;. &#xBCC0;&#xC885; &#xC0BD;&#xC785; &#xC0AC;&#xC774;&#xD2B8;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xAC00;&#xC871;&#xC73C;&#xB85C; &#xBD84;&#xB958; &#xB410;&#xB2E4; &#xD558; &#xACE0; &#xB192;&#xC740; &#xACC4;&#xC57D; &#xC774;&#xC804; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC;&#xC640; &#xACB0;&#xB860;&#xC744; &#xAD00;&#xCC30; &#xD588;&#xB2E4;: &#xB9E4;&#xD551;&#xD560; &#xB2E4;&#xB978; &#xC720;&#xAE30; &#xCCB4;&#xC5D0; &#xC0BD;&#xC785; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9;&#xB3C4; &#xC218;,&#xC774; &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; IS seq &#xBA54;&#xC11C;&#xB4DC; vivo&#xC5D0;&#xC11C; &#xC911;&#xB2E8;&#xB41C; loci&#xC758; &#xC774;&#xC804; &#xC870;&#xC0AC;&#xB97C; &#xD655;&#xC7A5; &#xD558; &#xACE0; &#xD504;&#xB85C; &#xBE59; M. &#xACB0;&#xD575; &#xAE34;&#xC7A5;&#xC5D0; &#xC911;&#xB2E8;&#xC758; &#xACB0;&#xACFC; &#xB300; &#xD55C; &#xCD08;&#xAE30; &#xACC4;&#xD68D;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

IS-seq: &#xC5EC;&#xB7EC; Mycobacterium &#xACB0;&#xD575; &#xC720;&#xC804;&#xC790;&#xC758; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; IS6110 &#xC804;&#xC704;&#xC758; &#xC0C8;&#xB85C;&#xC6B4; &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; &#xC870;&#xC0AC;.

Resumen: Antecedentes: el elemento de inserci&oacute;n IS6110 es una de las principales fuentes de variabilidad gen&oacute;mica en tuberculosis del Mycobacterium, el agente etiol&oacute;gico de la tuberculosis humana. Aunque IS6110 se ha utilizado ampliamente como marcador epidemiol&oacute;gico, la identificaci&oacute;n de los sitios de inserci&oacute;n cromos&oacute;micas precisa ha sido limitada por problemas t&eacute;cnicos. Aqu&iacute;, presentamos ISseq, un novedoso m&eacute;todo que combina la secuenciaci&oacute;n de alto rendimiento utilizando tecnolog&iacute;a de Illumina con eficiente muestra combinatoria multiplexaci&oacute;n para sonda simult&aacute;neamente 519 aislamientos cl&iacute;nicos, identificaci&oacute;n de casi todas las regiones que flanqueaban del elemento en un experimento &uacute;nico. Resultados: Se identificaron un total de 6.976 IS6110 regiones que flanqueaban en los diferentes aislamientos. Cuando valida utilizando cepas de referencia, el m&eacute;todo ten&iacute;a 100% especificidad y 98% valor predictivo positivo. Las inserciones asignadas a codificaci&oacute;n tanto no codificante regiones y en algunos casos interrumpido genes probablemente esencial para la virulencia o crecimiento in vitro. Las cepas se clasificaron en las familias usan sitios de inserci&oacute;n, y acuerdo alto con estudios previos se observ&oacute; conclusiones: este m&eacute;todo es-seq de alto rendimiento, que tambi&eacute;n puede utilizarse para asignar las inserciones en otros organismos, se extiende a encuestas anteriores de loci interrumpidos en vivo y proporciona una base para sondear las consecuencias de las interrupciones en cepas de M. tuberculosis.

IS-seq: un estudio de la novela de alto rendimiento de transposici&oacute;n de IS6110 in vivo en m&uacute;ltiples genomas de Mycobacterium tuberculosis.

Robi D. Mitra

Center for Genome Sciences and Systems Biology,
Department of Genetics,
Center for Genome Sciences and Systems Biology, Department of Genetics

Washington University School of Medicine

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

Robi D. Mitra

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Genome Sciences and Systems Biology,Department of Genetics,Center for Genome Sciences and Systems Biology, Department of Genetics

Washington University School of Medicine

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

Center for Genome Sciences and Systems Biology,
Department of Genetics,
Center for Genome Sciences and Systems Biology, Department of Genetics