Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

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Abstract

Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.

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The online version of this article (doi:10.1186/s13073-016-0375-z) contains supplementary material, which is available to authorized users.

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Most cited references 13

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Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

A Kallioniemi, D. Pinkel, D. Sudar … (1992)

Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. Analysis of tumor cell lines and primary bladder tumors identified 16 different regions of amplification, many in loci not previously known to be amplified.

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Array comparative genomic hybridization and its applications in cancer.

Donna Albertson, Daniel Pinkel (2005)

Alteration in DNA copy number is one of the many ways in which gene expression and function may be modified. Some variations are found among normal individuals, others occur in the course of normal processes in some species and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur before or shortly after fertilization, and DNA dosage-alteration changes occurring in somatic cells are frequent contributors to cancer. Detecting these aberrations and interpreting them in the context of broader knowledge facilitates the identification of crucial genes and pathways involved in biological processes and disease. Over the past several years, array comparative genomic hybridization has proven its value for analyzing DNA copy-number variations. Here, we discuss the state of the art of array comparative genomic hybridization and its applications in cancer, emphasizing general concepts rather than specific results.

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Accurate and comprehensive sequencing of personal genomes.

Geoff Parker, Hatice Ozel Abaan, Karin V Fuentes Fajardo … (2011)

As whole-genome sequencing becomes commoditized and we begin to sequence and analyze personal genomes for clinical and diagnostic purposes, it is necessary to understand what constitutes a complete sequencing experiment for determining genotypes and detecting single-nucleotide variants. Here, we show that the current recommendation of ∼30× coverage is not adequate to produce genotype calls across a large fraction of the genome with acceptably low error rates. Our results are based on analyses of a clinical sample sequenced on two related Illumina platforms, GAII(x) and HiSeq 2000, to a very high depth (126×). We used these data to establish genotype-calling filters that dramatically increase accuracy. We also empirically determined how the callable portion of the genome varies as a function of the amount of sequence data used. These results help provide a "sequencing guide" for future whole-genome sequencing decisions and metrics by which coverage statistics should be reported.

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Author and article information

Contributors

Kylie L. Gorringe: +613 85596521 , kylie.gorringe@petermac.org

Journal

Journal ID (nlm-ta): Genome Med

Journal ID (iso-abbrev): Genome Med

Title: Genome Medicine

Publisher: BioMed Central (London )

ISSN (Electronic): 1756-994X

Publication date (Electronic): 15 November 2016

Publication date PMC-release: 15 November 2016

Publication date Collection: 2016

Volume: 8

Electronic Location Identifier: 121

Affiliations

[1 ]Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC Australia

[2 ]The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC Australia

[3 ]Bioinformatics and Cancer Genomics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC Australia

[4 ]Molecular Biomarkers and Translational Genomics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC Australia

[5 ]LifePool, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC Australia

[6 ]Pathology, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC Australia

[7 ]Molecular Genomics Core Facility, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC Australia

[8 ]Molecular Imaging and Targeted Therapeutics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC Australia

[9 ]Department of Pathology, University of Melbourne, Parkville, VIC Australia

Article

Publisher ID: 375

DOI: 10.1186/s13073-016-0375-z

PMC ID: 5111221

PubMed ID: 27846907

SO-VID: ad6c9cd4-7f40-4762-bc61-091080f6592a

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 13 September 2016

Date accepted : 26 October 2016

Funding

Funded by: Australian National Health and Medical Research Council

Award ID: NHMRC APP1063092

Award Recipient : Kylie L. Gorringe

Funded by: FundRef http://dx.doi.org/10.13039/501100001026, National Breast Cancer Foundation;

Funded by: Peter MacCallum Cancer Foundation

Award ID: 1448

Funded by: NHMRC Early Career Fellowship

Award ID: APP1052904

Award Recipient : David L. Goode

Funded by: Melbourne International Research Scholarship

Funded by: Melbourne International Fee Remission Scholarship

Custom metadata

ScienceOpen disciplines: Molecular medicine

Keywords: low coverage whole genome sequencing (lc wgs),low-input dna,copy number,formalin-fixed paraffin-embedded (ffpe),next generation sequencing

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ScienceOpen disciplines: Molecular medicine

Keywords: low coverage whole genome sequencing (lc wgs), low-input dna, copy number, formalin-fixed paraffin-embedded (ffpe), next generation sequencing

Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

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Abstract

Electronic supplementary material

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Genome Engineering using CRISPR

Most cited references 13

Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

Array comparative genomic hybridization and its applications in cancer.

Accurate and comprehensive sequencing of personal genomes.

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