Agradecemos a M. Meyer para o conselho; A Academia Croata de Ciências e Artes e da Academia de Berlim-Brandenburgo de Ciências para o apoio logístico e científico, e do Fundo de Inovação Presidencial da Sociedade Max Planck para apoio financeiro. JMG foi apoiado por uma bolsa de pós-doutorado internacional NSF (OISE-0754461). Seqüências depositadas no banco de dados EBI de nucleotídeos com os números da adesão: Neandertal 1 (Feldhofer 1) FM865407, Neandertal 2 (Feldhofer 2) FM865408, Sidron 1253 FM865409, Vindija 33,25 FM865410, Mezmaiskaya um FM865411

Este método foi utilizado na pesquisa relatada em <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">Briggs et al. Ciência 325 (5938). 318-321 (2009)</a> . Reagentes necessários: <ul><li> AmpliTaq Polimerase DNA de Ouro </li><li> 10x GeneAmp PCR buffer II </li><li> MgCl 2 25mM </li><li> dNTP mix, 25 mM de cada </li><li> BSA, 10 mg ml-1 em água </li><li> Água de biologia molecular grau </li><li> EB buffer (fornecido com MinElute kit de purificação PCR), 10 mM Tris HCl, pH 8,5 </li><li> MinElute Kit de Purificação PCR </li><li> M-270 estreptavidina Dynabeads </li><li> 2x Encadernação e Wash (BW) tampão (2 M NaCl, 10 mM Tris-Cl, 1 mM EDTA, pH 8,0, 0,2% Tween 20) </li><li> 1x Encadernação e Tampão de lavagem (BW) (2x tampão 2X BindWash diluída em água) </li><li> Hot Wash (HW) buffer (2.5mm MgCl, 1X tampão Taq ouro, 0,1% Tween 20) </li></ul> Para informações sobre o fabricante quanto fora do padrão de reagentes e equipamentos mais tarde tabela. 1) ampliação da Biblioteca <ol><li> O modelo de DNA aqui é uma biblioteca de 454 preparados a partir de uma baixa fonte de DNA no número de cópias conforme descrito em (Rohland e Hofreiter, 2007a, 2007b) e (Maricic e Pääbo, 2009). Para amplificar toda a biblioteca, prepare a mistura PCR seguinte: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reagente </td><td> mL por reação </td></tr><tr><td> Água </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR buffer II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mM) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTPs (25 mM cada) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR primer/10uM rvs </td><td> 3 </td></tr><tr><td> AmpliTaq DNA polimerase Gold (5 U / mL) </td><td> 1 </td></tr><tr><td> 454 modelo de biblioteca de DNA </td><td> 25 </td></tr><tr><td> Total </td><td> 100 </td></tr></tbody></table></li><li> Use o programa de PCR a seguir em um cycler Thermo por 14 ciclos de amplificação <ol><li> 95 ° C 12min </li><li> 95 ° C 30 </li><li> 60 ° C (temperatura de recozimento ou desejado) 1 min </li><li> 72 ° C 1 min </li><li> Ir para 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Purificar a reação sobre uma coluna de spin Qiagen MinElute acordo com as instruções do fabricante. Eluir em 50 mL tampão EB. </li><li> Quantificar o produto purificado amplificado, bem como uma alíquota da biblioteca sem amplificação com qPCR (Meyer et al., 2007). Se o produto amplificado tem mais de 1 X 10 12 cópias por ul, reduzir a quantidade de modelo na etapa seguinte Primer Extension para garantir que não mais de 2 X 10 12 cópias são adicionados à reação de extensão Primer. </li></ol> 2) Extensão Primer <ol><li> Prepare uma mistura mestre para o número necessário de reações. <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> Reagente </td><td> mL por reação </td></tr><tr><td> Água </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR buffer II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mM) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTPs (25 mM cada) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR primer/10uM rvs </td><td> 3 </td></tr><tr><td> AmpliTaq DNA polimerase Gold (5 U / mL) </td><td> 1 </td></tr><tr><td> 454 modelo de biblioteca de DNA </td><td> 25 </td></tr><tr><td> Total </td><td> 100 </td></tr></tbody></table></li><li> Executar o programa a seguir em um Termociclador para a reação única cartilha extensão: <ol><li> 95 ° C 12min </li><li> 60 ° C (ou a sua cartilha PEC annealing temporário se for diferente) 1 min </li><li> 72 ° C 5 min </li><li> 72 ° C para sempre! Etapa crítica Mantenha reação após a extensão a 72 ° C. Em seguida, diretamente ou pipeta 150ul PBI PB buffer para QIAGEN purificação MinElute nos tubos antes de remover a partir do bloco de calor. Isto é importante para evitar a não-específicas de primer recozimento e captura como a mistura esfriar. </li></ol></li><li> Purificar a reação sobre uma coluna de spin Qiagen MinElute, de acordo com as instruções do fabricante. Eluir em 50 mL EB. </li></ol> 3) Captura de Produto Extensão <ol><li> Ressuspender solução estoque de M-270 contas em vortex. Retire 25 mL de suspensão talão por amostra. Lave as contas duas vezes com 500ul 2x BW buffer e ressuspender em 25 mL de buffer 2xBW por amostra. </li><li> Adicionar 25 mL de eluato passo 2,3-25 mL suspensão talão do passo 3.1. Misture em seguida, gire para 15 min à temperatura ambiente. Recomendações: manter restantes 5 ul do eluato a partir do passo 2.3 para quantificação qPCR. </li><li> Transfira a mistura toda para um novo tubo de 1,5 ml (o que ajuda a diminuir as transferências de fragmentos não-alvo da biblioteca). Pellet sobrenadante usando o coletor de partículas magnéticas (MPC) e desprezar o sobrenadante. </li><li> Lavar 5 vezes com 500 mL de buffer 1xBW (muitas lavagens ajudar a remover como pano de fundo tanto quanto possível). Após a etapa de lavagem passado, girar rapidamente e remover os últimos vestígios do sobrenadante. </li><li> Adicionar 500μl 1x Hot Tampão de lavagem (HW). Agitar durante 2 min a 65 ° C (ou 5C acima da cartilha PEC Tm) em um bloco térmico. Retire o sobrenadante rapidamente após isso, para minimizar a arrefecer. (Este passo é para remover fragmentos de fundo ainda associados com o PEC primers, mas não realmente estendido no durante a etapa de extensão). Remove os últimos vestígios do sobrenadante. </li><li> Ressuspender o sedimento talão em 30 mL tampão EB. Transfira a mistura toda para um novo tubo de 1,5 ml (o que ajuda a diminuir as transferências de fragmentos não-alvo da biblioteca). Incubar a 95 ° C por 3 minutos em um termociclador para a eluição. Lugar no MPC e remover o sobrenadante, tomando cuidado para deixar as contas para trás. </li></ol> 4) Captura de amplificação Produto <ol><li> Prepare uma mistura mestre PCR para o número necessário de amostras. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reagente </td><td> mL por reação </td></tr><tr><td> Água </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR buffer II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mM) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTPs (25 mM cada) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR primer/10uM rvs </td><td> 3 </td></tr><tr><td> AmpliTaq DNA polimerase Gold (5 U / mL) </td><td> 1 </td></tr><tr><td> Produto de captura eluída </td><td> 25 </td></tr><tr><td> Total </td><td> 100 </td></tr></tbody></table></li><li> Use o programa de PCR a seguir em um cycler Thermo por 14 ciclos de amplificação: <ol><li> 95 ° C 12min </li><li> 95 ° C 30 </li><li> 60 ° C (temperatura de recozimento ou desejado) 1 min </li><li> 72 ° C 1 min </li><li> Ir para 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Purificar a reação sobre uma coluna de spin Qiagen MinElute sílica de acordo com as instruções do fabricante. Eluir em 50 mL tampão EB. O produto pode ser usado agora, quer para uma segunda rodada de captação, começando no passo 2.1, ou entrou directamente para o 454 emulsão PCR protocolo, para o seqüenciamento. </li></ol> Resultados representativos: É altamente recomendável quando se inicia com o protocolo PEC para realizar uma reação de captura de primeira, onde uma pequena quantidade de uma &quot;seqüência alvo de controlo positivo&quot; (por exemplo, um produto da PCR ~ 100bp - 1 picograma é facilmente suficiente) é misturado com o normal recomendado quantidade de amplificado 454 modelo de biblioteca (veja o passo 2.1), e captura é feita com um primer PEC único projetado para capturar o produto controle positivo. Se essa reação controle é realizado qPCR, com tanto positivos específicos de controle e 454 adaptador específico pares de primers podem ser usados ​​para quantificar a quantidade de &#39;target controle&#39; versus fundo na reação purificada cartilha de extensão (1,3), produto de extensão de primer (2,3 ) e produto de captura eluída talão (3.6). Desta forma, tanto a eficácia da remoção de fundo (&quot;especificidade&quot;) ea eficiência de recuperação de destino (&quot;sensibilidade&quot;) em suas condições experimentais podem ser medidos diretamente. A bem sucedida PEC protocolo normalmente tem as seguintes propriedades - Sensibilidade (medido pela quantidade de alvo de controle mantidos através do protocolo): Montante total da meta de controle em produto de captura eluída talão (3.6) = ~ 10-20% do valor total em purificada reação de extensão de primer (2.3). Fundo (medido pelo par de primer emPCR 454): Montante total do fundo em produto de captura eluída talão (3.6) &lt;0,01% do montante total em reação purificada cartilha de extensão (2,3). Se houver menos de 1% da meta remanescentes no produto final, a etapa de extensão de primer pode ter sido mal sucedido e deve ser investigada. Se há muito mais do que 0,01% do fundo restantes nos produtos finais, os passos de lavagem pode ter sido incorrectamente realizada e deve ser investigada.

<ol>
	<li>Briggs, A. W., Good, J. M., Green, R. E., Krause, J., Maricic, T., Stenzel, U., Lalueza-Fox, C., Rudan, P., Brajkovic, D., Kucan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+retrieval+and+analysis+of+five+Neandertal+mtDNA+genomes.">Targeted retrieval and analysis of five Neandertal mtDNA genomes.</a> Science. 325 (5938), 318-321 (2009).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+optimization+of+ancient+DNA+extraction.">Comparison and optimization of ancient DNA extraction.</a> Biotechniques. 42 (3), 343-352 (2007).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+DNA+extraction+from+bones+and+teeth.">Ancient DNA extraction from bones and teeth.</a> Nat Protoc. 2 (7), 1756-1762 (2007).</li><li>Maricic, T., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+454+sequencing+library+preparation+from+small+amounts+of+DNA+permits+sequence+determination+of+both+DNA+strands.">Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.</a> Biotechniques. 46 (1), 51-57 (2009).</li><li>Meyer, M., AW, B. r. i. g. g. s., Maricic, T., Hober, B., Hoffner, B., Krause, J., Weihmann, A., Paabo, S., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+micrograms+to+picograms:+quantitative+PCR+reduces+the+material+demands+of+high-throughput+sequencing.">From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.</a> Nucleic Acids Res. 36 (1), e5-e5 (2007).</li></ol>

The authors have nothing to disclose.

O método PEC é simples, rápida, sensível e específico. Portanto, os autores prevêem múltiplas aplicações fora DNA antigo, como a captura de pequenos fragmentos de RNA de uma biblioteca de RNA, interrogatório de variação estrutural em uma amostra colectiva ou a captura de 16S (ou outros loci) a diversidade de uma amostra metagenomic. Um ponto a mencionar é que a sensibilidade da captura torna-se menor que o número de PEC primers em uma reação de multiplex aumenta captura. PEC não é, portanto, ideal para captura de regiões de captura muito grande (por exemplo megabase um ou mais), mas é extremamente adequado para a captura de regiões-alvo pequenas ou mesmo posições SNP de muitos indivíduos de uma forma rápida.

<table><tbody><tr><td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Applied Biosystems</td><td>N8080241</td><td></td></tr><tr><td>QIAGEN MinElute PCR purification kit</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>M-270 streptavidin beads</td><td>Invitrogen</td><td>653.05</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P2287</td><td></td></tr><tr><td>DynaMag-2 magnetic particle separator</td><td>Invitrogen</td><td>120.02D</td><td></td></tr></tbody></table>

primer extension capture: targeted sequence retrieval from heavily degraded dna sources

Nós apresentamos um método de recuperação de DNA alvo seqüência a partir de fontes de DNA que são altamente degradadas e contaminadas com DNA microbial, como é típico de ossos antigos. O método reduz destruição da amostra e as exigências em relação ao sequenciamento direto PCR ou abordagens seqüenciamento shotgun. Usamos este método para reconstruir a completa DNA mitocondrial (DNAmt) genomas de cinco neandertais de toda a sua abrangência geográfica. A diversidade genética do mtDNA neandertais final foi de aproximadamente três vezes menor do que o de humanos modernos contemporâneos. Juntamente com análises de mtDNA evolução de proteínas, estes dados sugerem que a longo prazo tamanho efetivo da população de neandertais era menor do que o de humanos modernos e os grandes símios existentes.

Watch this Scientific Journal Video about Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources at JoVE.com

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

Nós apresentamos um método de recuperação de seqüência de DNA alvo antigo, que usamos para reconstruir o genoma mitocondrial completo de cinco indivíduos Neandertal. Comparação dessas seqüências com os seres humanos hoje sugere que os neandertais tinham um longo prazo o tamanho da população de baixa eficácia.

Captura Primer Extensão: Recuperação Sequence alvejado a partir de Fontes de DNA altamente degradadas

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

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Research

JoVE Journal

Biology

20.1K visualizações.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. Nós apresentamos um método de recuperação de seqüência de DNA alvo antigo, que usamos para reconstruir o genoma mitocondrial completo de cinco indivíduos Neandertal. Comparação dessas seqüências com os seres humanos hoje sugere que os neandertais tinham um longo prazo o tamanho da população de baixa eficácia.

Vídeo: Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: Uma alta taxa de transferência baseada no sequenciamento técnica para identificar a tarde replicando regiões do genoma

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Genética

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

Adaptação da hibridação capturar de proteínas da cromatina associada para proteômica para células de mamíferos

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

Promotor captura Oi-c: Alta resolução, todo o genoma caracterização das interações do promotor

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Deteção de evento raro usando o DNA de correção de erro e a sequenciação do ARN

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

Identificação do elevado-throughput de sequências de genes regulatórios utilizando a próxima geração sequenciamento de conformação Circular cromossoma capturar (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Capturando a Conformação Cromossômica Através de Escalas de Comprimento

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

<meta charset="utf8"></head><body>Detectando eventos de clivagem CRISPR-Cas9 fora do alvo com CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

<meta charset="utf8"></head><body>CIRCLE-Seq para interrogatório de edição de genes fora do alvo

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

CIRCLE-Seq para interrogatório de edição de genes fora do alvo

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enriquecimento por uma tecnologia baseada em transposase para NGS Análise de toda a sequência de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt; e 9 genes envolvidos na reparação de danos no DNA

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Captura Primer Extensão: Recuperação Sequence alvejado a partir de Fontes de DNA altamente degradadas

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CIRCLE-Seq para interrogatório de edição de genes fora do alvo

CIRCLE-Seq para interrogatório de edição de genes fora do alvo

CIRCLE-Seq para interrogatório de edição de genes fora do alvo

gDNA Enriquecimento por uma tecnologia baseada em transposase para NGS Análise de toda a sequência de