Using Next-generation Sequencing for High Resolution Multiplex Analysis of Copy Number Variation from Nanogram Quantities of DNA from Formalin-fixed Paraffin-embedded Specimens

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2010 Jun 8

PMID 20525786

Citations 42

Authors

Henry M Wood

Ornella Belvedere

Caroline Conway

Catherine Daly

Rebecca Chalkley

Melissa Bickerdike

Claire McKinley

Phil Egan

Lisa Ross

Bruce Hayward

Joanne Morgan

Leslie Davidson

Ken Maclennan

Thian K Ong

Kostas Papagiannopoulos

Ian Cook

David J Adams

Graham R Taylor

Pamela Rabbitts

Affiliations

Soon will be listed here.

Abstract

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

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