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.

1. Assessment of gDNA (genomic DNA) Yield
<ol>
	<li>Quantify freshly extracted gDNA before preparation of the library. Use the fluorometric yield assessment method to quantify intact gDNA (avoid using a spectrophotometer for gDNA yield assessment). Measure the (260/280 nm) ratio and ensure that it is between 1.8 and 2. NOTE: 50 ng of gDNA are required for the experiment.</li>
</ol>
2. gDNA Enrichment: Day 1, Morning
<ol>
	<li>Before Starting
	<ol>
		<li>From the DNA enrichment kit (see Table o...

<ol>
	<li>Cornelisse, C. J., Cornelis, R. S., Devilee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+responsible+for+familial+breast+cancer.">Genes responsible for familial breast cancer.</a> Pathol. Res. Pract. 192 (7), 684-693 (1996).</li><li>Culver, J., Lowstuter, K., Bowling, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+breast+cancer+risk+and+BRCA1/2+carrier+probability.">Assessing breast cancer risk and BRCA1/2 carrier probability.</a> Breast Dis. 27, 5-20 (2007).</li><li>Cox, D. 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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. 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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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The widespread use of NGS devices and technologies has provided new opportunities in the study of cancer and genetic disorders. In addition to whole genome sequencing or RNA sequencing, the analysis of a large amount of selected gDNA sequences in numerous patients simultaneously offers great prospects in diagnosis. Here, we developed a specific design (available on demand) using Nextera technology to study 11 complete genes in 24 patients simultaneously with a medium throughput sequencing device (Table of Materials/Equip...

In 2010, nearly 1.5 million people (essentially women) developed breast cancer worldwide. It is estimated that 5 to 10% of these cases were hereditary. Almost 20 years ago, BRCA1 and BRCA2 were identified as involved in hereditary breast and ovarian cancers1. Since about 15 years ago, BRCA1 and BRCA2 coding regions have been sequenced to determine the genetic predisposition to breast and ovary cancer. Alterations in BRCA1 and BRCA2 are detected in 10 to 20% of selected families2 suggesting that the analysis of these regions is not sufficient for effective screening. Recently, the analysis of non-coding sequences (promoter, introns, 3-&#8217;UTR) of BRCA1 and BRCA2 highlighted that new mutations/variations could be linked to a higher risk of breast cancer3-6.
BRCA1 and BRCA2 proteins are involved in Homologous Recombination Repair (HHR), which is completed by numerous partners7. While alterations in BRCA1 or BRCA2 induce defects in DNA repair, the other partners may also affect the risk of breast cancer. This hypothesis appears to have been validated since BRIP18 and PALB29 have a proven impact on cervical and breast cancer, respectively. In addition, two other &#8220;moderate-risk&#8221; breast cancer susceptibility genes, ATM and CHEK2, may also be studied routinely10.
Following on from these studies, we decided to develop a protocol to analyze 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAP80, and TP53) in 24 patients simultaneously using a very easy and relatively fast protocol based on transposase technology, with enrichment and sequencing on a medium throughput device. Thanks to this technique, we sequenced complete genes from the start of the promoter to the end of 3&#8217;-UTR, except for RAP80, for which an intronic region of 2,500 bp was not covered (Chr5: 176,381,588-176,390,180). This represents a total of about 1,000,300 bp studied with 2,734 probes. Usually, BRCA1 and BRCA2 exonic sequences are analyzed by Sanger sequencing, which needs 1.5 months for less than 20 patients. With the present protocol (Figure 1), in the same time, 11 complete genes for more than 75 patients could be analyzed.

<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

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.

Sample QC Results
The ability of this method to determine sequences of target genes is based on the quality of the gDNA (Figure 2A) and the quality of the tagmentation step. If the tagmentation is not sufficient (Figure 2B, upper panel), the sequencing will not be satisfactory. As mentioned above, after the tagmentation purification, the gDNA should be tagmented into fragments from 150 bp to 1,000 bp with the majority of fragments around 300 bp (Figure 2B...

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

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 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 ...

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