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

Summary

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.

Abstract

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.

Introduction

The identification of "driver" 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 "next-generation" 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 ....

Protocol

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 extraction.

1. Aliquot 50-250 ng (250 ng recommended) of extracted DNA per sample, diluted in 1x Tris-EDTA (pH 8.0) buffer to a final volu....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NEBNext End Repair Module	New England Biolabs	E6050L
NEBNext dA-Tailing Module	New England Biolabs	E6053L
NEBNext Quick Ligation Module	New England Biolabs	E6056L
Agencourt AMPure XP	Beckman Coulter Genomics
NEXTflex PCR-Free Barcodes – 24	Bioo Scientific	514103
HiFi Library Amplification Kit	KAPA Biosystems	KK2612
COT Human DNA, Fluorometric Grade	Roche Diagnostics	05 480 647 001
NimbleGen SeqCap EZ Hybridization and Wash kit	Roche NimbleGen	05 634 261 001
SeqCap EZ Library Baits	Roche NimbleGen
QIAquick PCR Purification Kit	Qiagen	28104
Qubit dsDNA Broad Range (BR) Assay Kit	Life Technologies	Q32850
Qubit dsDNA High Sensitivity (HS) Assay Kit	Life Technologies	Q32851
Agilent DNA HS Kit	Agilent Technologies	5067-4626, 4627
Agilent 2100 Bioanalyzer	Agilent Technologies
Covaris E220	Covaris
Magnetic Stand-96	Ambion	AM10027
Illumina Hi-Seq 2000	Illumina