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A comprehensive multicenter comparison of whole genome sequencing pipelines using a uniform tumor-normal sample pair

Ivo Buchhalter, Barbara Hutter, Tyler S. Alioto, Timothy A. Beck, Paul C. Boutros, Benedikt Brors, Adam P. Butler, Sasithorn Chotewutmontri, Robert E. Denroche, Sophia Derdak, Nicolle Diessl, Lars Feuerbach, Akihiro Fujimoto, Susanne Gröbner, Marta Gut, Nicholas J. Harding, Michael Heinold, Lawrence E. Heisler, Jonathan Hinton, Natalie Jäger, David Jones, Rolf Kabbe, Andrey Korshunov, John D. McPherson, Andrew Menzies, Hidewaki Nakagawa, Christopher Previti, Keiran Raine, Paolo Ribeca, Sabine Schmidt, Rebecca Shepherd, Lucy Stebbings, Patrick S. Tarpey, Jon W. Teague, Laurie Tonon, David A. Wheeler, Liu Xi, Takafumi N. Yamaguchi, Anne-Sophie Sertier, Stefan M. Pfister, Peter J. Campbell, Matthias Schlesner, Peter Lichter, Roland Eils, Ivo G. Gut, David T. W. Jones, on behalf of the ICGC Verification and Validation Working Group
doi: https://doi.org/10.1101/013177
Ivo Buchhalter
1Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
2Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Barbara Hutter
2Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Tyler S. Alioto
3Centro Nacional de Análisis Genómico, Parc Científic de Barcelona, c/ Baldiri Reixac, 4-8, 08028 Barcelona, Spain
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Timothy A. Beck
4Ontario Institute for Cancer Research, 661 University Ave, Suite 510, Toronto Ontario Canada M5G 0A3
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Paul C. Boutros
4Ontario Institute for Cancer Research, 661 University Ave, Suite 510, Toronto Ontario Canada M5G 0A3
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Benedikt Brors
2Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Adam P. Butler
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Sasithorn Chotewutmontri
6Genome and Proteome Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Robert E. Denroche
4Ontario Institute for Cancer Research, 661 University Ave, Suite 510, Toronto Ontario Canada M5G 0A3
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Sophia Derdak
3Centro Nacional de Análisis Genómico, Parc Científic de Barcelona, c/ Baldiri Reixac, 4-8, 08028 Barcelona, Spain
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Nicolle Diessl
6Genome and Proteome Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Lars Feuerbach
2Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Akihiro Fujimoto
7RIKEN Center for Integrative Medical Sciences, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Susanne Gröbner
8Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Im Neuenheimer Feld 430, 69120, Heidelberg, Germany
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Marta Gut
3Centro Nacional de Análisis Genómico, Parc Científic de Barcelona, c/ Baldiri Reixac, 4-8, 08028 Barcelona, Spain
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Nicholas J. Harding
4Ontario Institute for Cancer Research, 661 University Ave, Suite 510, Toronto Ontario Canada M5G 0A3
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Michael Heinold
2Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Lawrence E. Heisler
4Ontario Institute for Cancer Research, 661 University Ave, Suite 510, Toronto Ontario Canada M5G 0A3
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Jonathan Hinton
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Natalie Jäger
1Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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David Jones
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Rolf Kabbe
1Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Andrey Korshunov
9Department of Neuropathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany
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John D. McPherson
4Ontario Institute for Cancer Research, 661 University Ave, Suite 510, Toronto Ontario Canada M5G 0A3
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Andrew Menzies
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Hidewaki Nakagawa
7RIKEN Center for Integrative Medical Sciences, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Christopher Previti
6Genome and Proteome Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Keiran Raine
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Paolo Ribeca
3Centro Nacional de Análisis Genómico, Parc Científic de Barcelona, c/ Baldiri Reixac, 4-8, 08028 Barcelona, Spain
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Sabine Schmidt
6Genome and Proteome Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Rebecca Shepherd
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Lucy Stebbings
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Patrick S. Tarpey
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Jon W. Teague
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Laurie Tonon
10Synergie Lyon Cancer Foundation, Centre Léon Bérard, Cheney C, 28 rue Laennec, 69373 LYON Cedex 08
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David A. Wheeler
11Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, U.S.A.
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Liu Xi
11Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, U.S.A.
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Takafumi N. Yamaguchi
4Ontario Institute for Cancer Research, 661 University Ave, Suite 510, Toronto Ontario Canada M5G 0A3
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Anne-Sophie Sertier
10Synergie Lyon Cancer Foundation, Centre Léon Bérard, Cheney C, 28 rue Laennec, 69373 LYON Cedex 08
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Stefan M. Pfister
8Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Im Neuenheimer Feld 430, 69120, Heidelberg, Germany
12Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Peter J. Campbell
5Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, CB10 1SA
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Matthias Schlesner
1Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Peter Lichter
13Division of Molecular Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Roland Eils
1Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
14Institute of Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Heidelberg, 69120, Germany
15Bioquant Center, University of Heidelberg, Im Neuenheimer Feld 267, Heidelberg, 69120, Germany
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Ivo G. Gut
3Centro Nacional de Análisis Genómico, Parc Científic de Barcelona, c/ Baldiri Reixac, 4-8, 08028 Barcelona, Spain
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David T. W. Jones
8Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Im Neuenheimer Feld 430, 69120, Heidelberg, Germany
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Abstract

As next-generation sequencing becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Through the International Cancer Genome Consortium (ICGC), we compared sequencing pipelines at five independent centers (CNAG, DKFZ, OICR, RIKEN and WTSI) using a single tumor-blood DNA pair. Analyses by each center and with one standardized algorithm revealed significant discrepancies. Although most pipelines performed well for coding mutations, library preparation methods and sequencing coverage metrics clearly influenced downstream results. PCR-free methods showed reduced GCbias and more even coverage. Increasing sequencing depth to ~100x (about three times current standards) showed a benefit, as long as the tumor: control coverage ratio remained balanced. To become part of routine clinical care, high-throughput sequencing must be globally compatible and comparable. This benchmarking exercise has highlighted several fundamental parameters to consider in this regard, which will allow for better optimization and planning of both basic and translational studies.

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Posted December 24, 2014.
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A comprehensive multicenter comparison of whole genome sequencing pipelines using a uniform tumor-normal sample pair
Ivo Buchhalter, Barbara Hutter, Tyler S. Alioto, Timothy A. Beck, Paul C. Boutros, Benedikt Brors, Adam P. Butler, Sasithorn Chotewutmontri, Robert E. Denroche, Sophia Derdak, Nicolle Diessl, Lars Feuerbach, Akihiro Fujimoto, Susanne Gröbner, Marta Gut, Nicholas J. Harding, Michael Heinold, Lawrence E. Heisler, Jonathan Hinton, Natalie Jäger, David Jones, Rolf Kabbe, Andrey Korshunov, John D. McPherson, Andrew Menzies, Hidewaki Nakagawa, Christopher Previti, Keiran Raine, Paolo Ribeca, Sabine Schmidt, Rebecca Shepherd, Lucy Stebbings, Patrick S. Tarpey, Jon W. Teague, Laurie Tonon, David A. Wheeler, Liu Xi, Takafumi N. Yamaguchi, Anne-Sophie Sertier, Stefan M. Pfister, Peter J. Campbell, Matthias Schlesner, Peter Lichter, Roland Eils, Ivo G. Gut, David T. W. Jones, on behalf of the ICGC Verification and Validation Working Group
bioRxiv 013177; doi: https://doi.org/10.1101/013177
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A comprehensive multicenter comparison of whole genome sequencing pipelines using a uniform tumor-normal sample pair
Ivo Buchhalter, Barbara Hutter, Tyler S. Alioto, Timothy A. Beck, Paul C. Boutros, Benedikt Brors, Adam P. Butler, Sasithorn Chotewutmontri, Robert E. Denroche, Sophia Derdak, Nicolle Diessl, Lars Feuerbach, Akihiro Fujimoto, Susanne Gröbner, Marta Gut, Nicholas J. Harding, Michael Heinold, Lawrence E. Heisler, Jonathan Hinton, Natalie Jäger, David Jones, Rolf Kabbe, Andrey Korshunov, John D. McPherson, Andrew Menzies, Hidewaki Nakagawa, Christopher Previti, Keiran Raine, Paolo Ribeca, Sabine Schmidt, Rebecca Shepherd, Lucy Stebbings, Patrick S. Tarpey, Jon W. Teague, Laurie Tonon, David A. Wheeler, Liu Xi, Takafumi N. Yamaguchi, Anne-Sophie Sertier, Stefan M. Pfister, Peter J. Campbell, Matthias Schlesner, Peter Lichter, Roland Eils, Ivo G. Gut, David T. W. Jones, on behalf of the ICGC Verification and Validation Working Group
bioRxiv 013177; doi: https://doi.org/10.1101/013177

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