Name: detecting somatic genetic alterations in tumor specimens by exon capture and massively parallel sequencing
Uploaded: 2013-10-18T19:00:00
Description: Watch this Scientific Journal Video about Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing at JoVE.com

We thank Dr. Agnes Viale and the MSKCC Genomics Core Laboratory for technical assistance. This protocol was developed with support from the Geoffrey Beene Cancer Research Center and the Farmer Family Foundation.

1. DNA and Reagent Preparation
Note: This protocol describes the simultaneous processing and analysis of 24 samples (e.g. 12 tumor/normal pairs) but can be adapted for smaller and larger batches. DNA samples may derive from FFPE or fresh frozen tissue, cytological specimens, or blood. Typically, both tumor and normal tissue from the same patient will be profiled together in order to distinguish somatic mutations from inherited polymorphisms. The protocol begins immediately following DNA...

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Our IMPACT assay produces a high alignment rate, high on-target rate, high target coverage, and high sensitivity for detecting mutations, indels, and copy number alterations. We have demonstrated the capability of our IMPACT assay to sequence DNA from both fresh frozen and archived FFPE samples of low DNA input. By performing targeted exon sequencing of key cancer-associated genes, one can achieve very deep sequence coverage for the exons of these most critical genes thereby maximizing the ability to detect low frequency...

The identification of &#34;driver&#34; tumor genetic events in key oncogenes and tumor suppressor genes plays an essential role in the diagnosis and treatment of many cancers1. Large-scale research efforts utilizing massively parallel &#34;next-generation&#34; sequencing have enabled the identification of many such cancer-associated genes in recent years2. However, these sequencing platforms typically require large quantities of DNA isolated from fresh frozen tissues, thus posing a major limitation in characterizing and analyzing DNA mutations from preserved tissues, such as formalin-fixed paraffin embedded (FFPE) tumor samples. Improved efforts to efficiently and reliably characterize &#34;actionable&#34; genomic information from FFPE tumor samples will enable the retrospective analysis of previously-banked specimens and further encourage individualized approaches to cancer management.
Traditionally, molecular diagnostic laboratories have relied on time-consuming, low-throughput methodologies such as Sanger sequencing and real-time PCR for DNA mutation profiling. More recently, higher-throughput methods utilizing multiplexed PCR or mass spectrometric genotyping have been developed to investigate recurrent somatic mutations in key cancer genes3-5. These approaches, however, are limited in that only predesignated &#34;hotspot&#34; mutations are assayed, making them unsuitable for detecting inactivating mutations in tumor suppressor genes. Massively parallel sequencing offers several advantages over these strategies including the ability to interrogate entire exons for both common and rare mutations, the ability to reveal additional classes of genomic alterations such as copy number gains and losses, and greater detection sensitivity in heterogeneous samples6, 7. Whole genome sequencing represents the most comprehensive approach for mutation discovery, though it is relatively expensive and incurs large computational demands for data analysis and storage.
For clinical applications, where only a small fraction of the genome may be of clinical interest, two particular innovations in sequencing technology have been transformative. First, through hybridization-based exon capture, one can isolate DNA corresponding to key cancer-associated genes for targeted mutation profiling8, . Second, through ligation of molecular barcodes (i.e. DNA sequences 6-8 nucleotides in length), one can pool hundreds of samples per sequencing run and fully take advantage of the ever-increasing capacity of massively parallel sequencing instruments10. When combined, these innovations enable tumors to be profiled for lower cost and at higher throughput, with smaller computational requirements11. Further, by redistributing sequence coverage to only those genes most critical to the particular application, one can achieve greater sequencing depth for higher detection sensitivity for low allele frequency events.
Here we describe our IMPACT assay (Integrated Mutation Profiling of Actionable Cancer Targets), which utilizes exon capture on barcoded sequence library pools by hybridization using custom oligonucleotides to capture all protein-coding exons and select introns of 279 key cancer-associated genes (Table 1). This strategy enables the identification of mutations, indels, copy number alterations, and select structural rearrangements involving these 279 genes. Our method is compatible with DNA isolated from both fresh frozen and FFPE tissue as well as fine needle aspirates and other cytology specimens.

<table border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody> 
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext End Repair Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6050L</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext dA-Tailing Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6053L</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext Quick Ligation Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6056L</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agencourt AMPure XP</td>
			<td style="width:247px;">Beckman Coulter Genomics</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEXTflex PCR-Free Barcodes &#8211; 24</td>
			<td style="width:247px;">Bioo Scientific</td>
			<td style="width:137px;">514103</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HiFi Library Amplification Kit</td>
			<td style="width:247px;">KAPA Biosystems</td>
			<td style="width:137px;">KK2612</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">COT Human DNA, Fluorometric Grade</td>
			<td style="width:247px;">Roche Diagnostics</td>
			<td style="width:137px;">05 480 647 001</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NimbleGen SeqCap EZ Hybridization and Wash kit</td>
			<td style="width:247px;">Roche NimbleGen</td>
			<td style="width:137px;">05 634 261 001</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">SeqCap EZ Library Baits</td>
			<td style="width:247px;">Roche NimbleGen</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">QIAquick PCR Purification Kit</td>
			<td style="width:247px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Qubit dsDNA Broad Range (BR) Assay Kit</td>
			<td style="width:247px;">Life Technologies</td>
			<td style="width:137px;">Q32850</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Qubit dsDNA High Sensitivity (HS) Assay Kit</td>
			<td style="width:247px;">Life Technologies</td>
			<td style="width:137px;">Q32851</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agilent DNA HS Kit</td>
			<td style="width:247px;">Agilent Technologies</td>
			<td style="width:137px;">5067-4626, 4627</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agilent 2100 Bioanalyzer</td>
			<td style="width:247px;">Agilent Technologies</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Covaris E220</td>
			<td style="width:247px;">Covaris</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Magnetic Stand-96</td>
			<td style="width:247px;">Ambion</td>
			<td style="width:137px;">AM10027</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Illumina Hi-Seq 2000</td>
			<td style="width:247px;">Illumina</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
	</tbody>
</table>

detecting somatic genetic alterations in tumor specimens by exon capture and massively parallel sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

One pool of 24 barcoded sequence libraries (12 tumor-normal pairs) was captured using probes corresponding to all protein-coding exons of 279 cancer genes and sequenced as 2 x 75 bp reads on a single lane of a HiSeq 2000 flow cell. Tumor and normal libraries were pooled in a 2:1 ratio. Sample performance metrics for a pool of frozen tumor DNA samples are shown in Figure 1, including alignment rate, fragment size distribution, on-target capture specificity, and mean target coverage. Example somatic mutati...

Watch this Scientific Journal Video about Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing at JoVE.com

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

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