title: DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly.
abstract: Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures. In practice, very useful profiles can be obtained with ∼0.1× genome coverage. We improve on previous methods by first implementing a combined correction for sequence mappability and GC content, and second, by applying this procedure to sequence data from the 1000 Genomes Project in order to develop a blacklist of problematic genome regions. A small subset of these blacklisted regions was previously identified by ENCODE, but the vast majority are novel unappreciated problematic regions. Our procedures are implemented in a pipeline called QDNAseq. We have analyzed over 1000 samples, most of which were obtained from the fixed tissue archives of more than 25 institutions. We demonstrate that for most samples our sequencing and analysis procedures yield genome profiles with noise levels near the statistical limit imposed by read counting. The described procedures also provide better correction of artifacts introduced by low DNA quality than prior approaches and better copy number data than high-resolution microarrays at a substantially lower cost.
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands; Department of Pathology, Haartman Institute and HUSLAB, FIN-00014 University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland;
authors.forename: Ilari
authors.initials: I
authors.lastname: Scheinin
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands;
authors.forename: Daoud
authors.initials: D
authors.lastname: Sie
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94158, USA; Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California 94158, USA;
authors.forename: Henrik
authors.initials: H
authors.lastname: Bengtsson
authors.affiliation: Department of Epidemiology and Biostatistics, VU University Medical Center, 1007 MB Amsterdam, The Netherlands; Department of Mathematics, VU University, 1181 HV Amsterdam, The Netherlands;
authors.forename: Mark A
authors.initials: MA
authors.lastname: van de Wiel
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94158, USA; Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California 94158, USA;
authors.forename: Adam B
authors.initials: AB
authors.lastname: Olshen
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands; Department of Neurology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands;
authors.forename: Hinke F
authors.initials: HF
authors.lastname: van Thuijl
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands;
authors.forename: Hendrik F
authors.initials: HF
authors.lastname: van Essen
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands;
authors.forename: Paul P
authors.initials: PP
authors.lastname: Eijk
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands;
authors.forename: François
authors.initials: F
authors.lastname: Rustenburg
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands;
authors.forename: Gerrit A
authors.initials: GA
authors.lastname: Meijer
authors.affiliation: Department of Neurology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands; Department of Neurology, Academic Medical Centre, 1105 AZ Amsterdam, The Netherlands;
authors.forename: Jaap C
authors.initials: JC
authors.lastname: Reijneveld
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands; Department of Pathology, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands;
authors.forename: Pieter
authors.initials: P
authors.lastname: Wesseling
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94158, USA; Department of Laboratory Medicine, University of California San Francisco, San Francisco, California 94153, USA;
authors.forename: Daniel
authors.initials: D
authors.lastname: Pinkel
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94158, USA; Department of Laboratory Medicine, University of California San Francisco, San Francisco, California 94153, USA; Bluestone Center for Clinical Research, New York University College of Dentistry, New York, New York 10010-4086, USA.
authors.forename: Donna G
authors.initials: DG
authors.lastname: Albertson
authors.affiliation: Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands; B.Ylstra@vumc.nl.
authors.forename: Bauke
authors.initials: B
authors.lastname: Ylstra
mesh_terms: D000465:Algorithms; D001482:Base Composition; D045744:Cell Line, Tumor; D055028:Comparative Genomic Hybridization; D019295:Computational Biology / Q000379:methods; D056915:DNA Copy Number Variations; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D012984:Software