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 (Video) | JoVE Journal
Uploaded: 2014-10-06T13:00:00.000+00:00
Duration: 8 min 15 s
Description: Learn transposase-based gDNA enrichment for NGS analysis of 11 DNA damage repair genes including BRCA1 and BRCA2. This efficient method enables simultaneous sequencing in under two weeks.

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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.

<ol><li>Cornelisse, C. J., Cornelis, R. S., Devilee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genes+responsible+for+familial+breast+cancer%5BTitle%5D)+AND+%22Pathol.+Res.+Pract%22%5BJournal%5D)">Genes responsible for familial breast cancer.</a> Pathol. Res. Pract. 192 (7), 684-693 (1996).</li>
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<li>Anczuków, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BRCA2+deep+intronic+mutation+causing+activation+of+a+cryptic+exon%3A+opening+toward+a+new+preventive+therapeutic+strategy%5BTitle%5D)+AND+%22Clin.+Cancer+Res%22%5BJournal%5D)">BRCA2 deep intronic mutation causing activation of a cryptic exon: opening toward a new preventive therapeutic strategy.</a> Clin. Cancer Res. 18 (18), 4903-4909 (2012).</li>
<li>Brewster, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+fifteen+novel+germline+variants+in+the+BRCA1+3%27UTR+reveals+a+variant+in+a+breast+cancer+case+that+introduces+a+functional+miR-103+target+site%5BTitle%5D)+AND+%22Hum.+Mutat%22%5BJournal%5D)">Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site.</a> Hum. Mutat. 33 (12), 1665-1675 (2012).</li>
<li>Roy, R., Chun, J., Powell, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BRCA1+and+BRCA2%3A+different+roles+in+a+common+pathway+of+genome+protection%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">BRCA1 and BRCA2: different roles in a common pathway of genome protection.</a> Nat Rev Cancer. 12 (1), 68-78 (2012).</li>
<li>Ma, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+evidence+for+the+contribution+of+the+genetic+variations+of+BRCA1-interacting+protein+1+%28BRIP1%29+to+the+genetic+susceptibility+of+cervical+cancer%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">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>Haanpää, M., Pylkäs, K., Moilanen, J. S., Winqvist, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+need+for+routine+clinical+testing+of+PALB2+c.1592delT+mutation+in+BRCA+negative+Northern+Finnish+breast+cancer+families%5BTitle%5D)+AND+%22BMC+Med.+Genet%22%5BJournal%5D)">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/pubmed?term=((PALB2+and+breast+cancer%3A+ready+for+clinical+translation%5BTitle%5D)+AND+%22Appl.+Clin.+Genet%22%5BJournal%5D)">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/pubmed?term=((Technical+and+implementation+issues+in+using+next-generation+sequencing+of+cancers+in+clinical+practice%5BTitle%5D)+AND+%22Br.+J.+Cancer%22%5BJournal%5D)">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., Borràs, E., de Sousa Dias, M., Gamundi, M. J., Mañé, B., Llort, G., Agúndez, J. A., Blanca, M., Carballo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+genomic+variations+in+BRCA1+and+BRCA2+genes+by+long-range+PCR+and+next-generation+sequencing%5BTitle%5D)+AND+%22J.+Mol.+Diagn%22%5BJournal%5D)">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/pubmed?term=((Systematic+comparison+of+three+methods+for+fragmentation+of+long-range+PCR+products+for+next+generation+sequencing%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">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., Borràs, E., Mañé, B., Gamundi, M. J., Carballo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+novel+mutations+that+cause+autosomal+dominant+retinitis+pigmentosa+in+candidate+genes+by+long-range+PCR+amplification+and+next-generation+sequencing%5BTitle%5D)+AND+%22Mol.+Vis%22%5BJournal%5D)">Detection of novel mutations that cause autosomal dominant retinitis pigmentosa in candidate genes by long-range PCR amplification and next-generation sequencing.</a> Mol. Vis. 19, 654-664 (2013).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Breast+Cancer+Linkage+Consortium.+Cancer+Risks+in+BRCA2+Mutation+Carriers%5BTitle%5D)+AND+%22J.+Natl.+Cancer+Inst%22%5BJournal%5D)">The Breast Cancer Linkage Consortium. Cancer Risks in BRCA2 Mutation Carriers.</a> J. Natl. Cancer Inst. 91, 1310-1316 (1999).</li>
<li>Levy-Lahad, E., Friedman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+risks+among+BRCA1+and+BRCA2+mutation+carriers%5BTitle%5D)+AND+%22Br.+J.+Cancer%22%5BJournal%5D)">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/pubmed?term=((Population+BRCA1+and+BRCA2+mutation+frequencies+and+cancer+penetrances%3A+a+kin-cohort+study+in+Ontario%2C+Canada%5BTitle%5D)+AND+%22J.+Natl.+Cancer+Inst%22%5BJournal%5D)">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>
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</ol>

The authors have no conflicts of interest to declare.

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><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&lt;br/&gt; Experiment Manager software&lt;br/&gt; Internet adress: http://designstudio.illumina.com/NexteraRc/project/new&lt;br/&gt; 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

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...

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 ...

gdna-enrichment-transposase-based-technology-for-ngs-analysis-whole

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Biology

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described.  The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues.  Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site—in essence, analyzing onsite mutagenesis.

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Whole Mount in situ Hybridization of Early Chick Embryos

Learn transposase-based gDNA enrichment for NGS analysis of 11 DNA damage repair genes including BRCA1 and BRCA2. This efficient method enables simultaneous sequencing in under two weeks.

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 (Video) | JoVE Journal

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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