Name: 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
Uploaded: 2014-10-06T13:00:00
Description: Watch this Scientific Journal Video about 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 at JoVE.co

We thank the Ligue contre le Cancer de C&#244;te d&#8217;Or and the Centre Georges-Fran&#231;ois Leclerc for their financial support. We thank Philip Bastable for the editing of the manuscript.

基因组DNA 1。评估（基因组DNA）产量<ol><li>量化新鲜提取基因组DNA的制备图书馆前。使用荧光估产方法量化完整的基因组DNA（避免使用分光光度计进行基因组DNA的产量评估）。测量（二百八十零分之二百六十nm）的比率，并确保它是在1.8和2注：50纳克基因组DNA的需要进行实验。 </li></ol> 2，基因组DNA富集：第1天，上午<ol><li>开始之前<ol><li>从DNA富集试剂盒（见表材料/设备），解?...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+evidence+for+the+contribution+of+the+genetic+variations+of+BRCA1-interacting+protein+1+(BRIP1)+to+the+genetic+susceptibility+of+cervical+cancer.">First evidence for the contribution of the genetic variations of BRCA1-interacting protein 1 (BRIP1) to the genetic susceptibility of cervical cancer.</a> Gene. 524 (2), 208-213 (2013).</li><li>Haanp&#228;&#228;, M., Pylk&#228;s, K., Moilanen, J. S., Winqvist, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+need+for+routine+clinical+testing+of+PALB2+c.1592delT+mutation+in+BRCA+negative+Northern+Finnish+breast+cancer+families.">Evaluation of the need for routine clinical testing of PALB2 c.1592delT mutation in BRCA negative Northern Finnish breast cancer families.</a> BMC Med. Genet. 14, 82 (2013).</li><li>Southey, M. C., Teo, Z. L., Winship, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PALB2+and+breast+cancer:+ready+for+clinical+translation.">PALB2 and breast cancer: ready for clinical translation.</a> Appl. Clin. Genet. 6, 43-52 (2013).</li><li>Ulahannan, D., Kovac, M. B., Mulholland, P. J., Cazier, J. B., Tomlinson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+and+implementation+issues+in+using+next-generation+sequencing+of+cancers+in+clinical+practice.">Technical and implementation issues in using next-generation sequencing of cancers in clinical practice.</a> Br. J. Cancer. 109, 827-835 (2013).</li><li>Hernan, I., Borr&#224;s, E., de Sousa Dias, M., Gamundi, M. J., Ma&#241;&#233;, B., Llort, G., Ag&#250;ndez, J. A., Blanca, M., Carballo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+genomic+variations+in+BRCA1+and+BRCA2+genes+by+long-range+PCR+and+next-generation+sequencing.">Detection of genomic variations in BRCA1 and BRCA2 genes by long-range PCR and next-generation sequencing.</a> J. Mol. Diagn. 14, 286-293 (2012).</li><li>Knierim, E., Lucke, B., Schwarz, J. M., Schuelke, M., Seelow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+three+methods+for+fragmentation+of+long-range+PCR+products+for+next+generation+sequencing.">Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.</a> Plos One. 6, e28240 (2011).</li><li>de Sousa Dias, M., Hernan, I., Pascual, B., Borr&#224;s, E., Ma&#241;&#233;, B., Gamundi, M. 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Cancer Inst. 91, 1310-1316 (1999).</li><li>Levy-Lahad, E., Friedman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+risks+among+BRCA1+and+BRCA2+mutation+carriers.">Cancer risks among BRCA1 and BRCA2 mutation carriers.</a> Br. J. Cancer. 96 (1), 11-15 (2007).</li><li>Risch, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+BRCA1+and+BRCA2+mutation+frequencies+and+cancer+penetrances:+a+kin-cohort+study+in+Ontario,+Canada.">Population BRCA1 and BRCA2 mutation frequencies and cancer penetrances: a kin-cohort study in Ontario, Canada.</a> J. Natl. Cancer Inst. 98 (23), 1694-1706 (2006).</li><li>Slater, E. 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广泛使用的NGS的设备和技术已经在癌症和遗传疾病的研究提供新的机会。除了全基因组测序和RNA测序，产生大量的无数的患者选择的基因组DNA序列的分析同时提供了诊断的前景十分广阔。在这里，我们开发了使用Nextera技术，同时研究11完整的基因在24例中等通量测序装置（表材料/设备）的特定设计（随叫随到）。该协议允许快速生成的数据，使患者的错误的低风险的担忧快速反应。如示于?...

2010年，有近150万人（主要是女性）开发的全球乳腺癌。据估计，5％至10％的这些情况是遗传的。大约20年前，BRCA1和BRCA2被认定为参与遗传性乳腺癌和卵巢癌1。自从大约15年前，BRCA1和BRCA2基因的编码区已被测序，以确定遗传倾向的乳腺癌和卵巢癌。在BRCA1和BRCA2的变化在10％至20％选家庭2表明这些区域的分析是不够有效的筛查检测。近日，BRCA1和BRCA2基因的非编码序列（启动子，内含子，3&#39;UTR）的分析，强调了新的基因突变/变异可能与乳腺癌3-6的风险较高。 BRCA1和BRCA2蛋白参与同源重组修复（HHR），它是由众多的合作伙伴7完成。而在BRCA1或BRCA2基因的改变诱发的DNA修复缺陷，其他伙伴也可能会影响到乳腺癌的危险性。这个假设似乎因为BRIP1 8已被验证，PALB2 9对宫颈癌和乳腺癌的一个行之有效的影响，分别为。此外，还有两个其他的“中度风险”的乳腺癌易感基因，ATM和CHEK2，也可以定期研究10。 从这些研究之后，我们决定开发一个协议分析11个基因（ 自动取款机，BARD1，BRCA1，BRCA2，BRIP1，CHEK2，PALB2，RAD50，RAD51C，RAP80和TP53），24例同时使用一个非常简单且相对基于转座技术快速的协议，以充实和测序中等吞吐量的设备上。谢谢该技术，我们测序完成基因的启动子的3&#39;-UTR的端部的起动，除了RAP80，对于其中的2500 bp的内含子区域没有被覆盖的（CHR 5：176,381,588-176,390,180）。这代表共约1000300 BP研究与2,734探头。通常，BRCA1和BRCA2的外显子序列由Sanger测序，这需要1.5个月时间少于20名患者进行分析。本协议（ 图1），在相同的时间，11个完整的基因为大于75的病人可以进行分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MiSeq</td>
			<td>Illumina</td>
			<td>SY-410-1001</td>
			<td>Sequencing/medium throughput device</td>
		</tr>
		<tr>
			<td>Nextera Enrichment kit</td>
			<td>Illumina</td>
			<td>FC-123-1208</td>
			<td>Transposase based technology</td>
		</tr>
		<tr>
			<td>300 cycle cartridge</td>
			<td>Illumina</td>
			<td>15033624</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Magnetic purification beads</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Alpaqua</td>
			<td>A32782</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Life Technologies</td>
			<td>4306737</td>
			<td></td>
		</tr>
		<tr>
			<td>MIDI 96-well plates</td>
			<td>Biorad</td>
			<td>AB0859</td>
			<td></td>
		</tr>
		<tr>
			<td>Microseal A</td>
			<td>Biorad</td>
			<td>MSA-5001</td>
			<td>This seal is necessary only for PCR amplification. Other standard seals can be used throughout the experiment</td>
		</tr>
		<tr>
			<td>MiSeq</td>
			<td>Illumina</td>
			<td></td>
			<td>Provided with the sequencing device 
			Experiment Manager software</td>
		</tr>
		<tr>
			<td></td>
			<td>Illumina</td>
			<td></td>
			<td>Internet adress: http://designstudio.illumina.com/NexteraRc/project/new&#62; 
			Manufacturer website tool</td>
		</tr>
	</tbody>
</table>

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

广泛使用的新一代测序开辟了为癌症研究和诊断的新途径。农工商会带来癌症大量的新数据，尤其是癌症遗传学。目前的知识和未来发现将使得有必要研究一种巨大的基因可能涉及遗传因素对癌症的数目。在这方面，我们开发了一个Nextera设计研究11参与DNA损伤修复完整的基因。该协议的开发是为了安全地同时学习11个基因（ATM，BARD1，BRCA1，BRCA2，BRIP1，CHEK2，PALB2，RAD50，RAD51C，RAD80和TP53）从启动到3&#39;-UTR 24例。这个协议中，根据转座技术和基因组DNA富集，给出了一个很大的优点的时间为基因诊断由于采样复用条件。这个协议可以安全地使用与血液的gDNA。

样品QC成果这种方法来确定靶基因的序列的能力是基于基因组DNA（ 图2A）的质量和tagmentation步骤的质量。如果tagmentation是不够（ 图2B，上图），测序将不会令人满意。如上面所提到的，tagmentation纯化后，将基因组DNA的应tagmented成片段从150 100bp到1000bp的多数大约300 bp的片段（ 图2B，下图）。 在文库制备结束，库质量是通?...

Watch this Scientific Journal Video about 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 at JoVE.co

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

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

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.

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 ...

Research

JoVE Journal

Biology

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

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