RESEARCH LETTER variations were identified using BreakDancer.All predicted non-repetitive so- 18.Ding,L.et al.Somatic mutations affect key pathways in lung adenocarcinoma. matic SNVs,indels and all structural variants were included on custom sequence re455,1069-1075(2008) capture arrays from Roche Nimblegen.Illumina 2 X 100-bp paired-end sequen- 19.Mullighan,C.G.et al.Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia.Science 322,1377-1380(2008). cing reads were produced after elution from capture arrays.VarScan and a read 20.Anderson,K.etal Genetic variegation of clonal architecture and propagating cells remapping strategy using Crossmatch(P.Green,unpublished data)and BWA in leukaemia.Nature 469.356-361 (2011). were used for determining the validation status of predicted SNVs,indels and 21.Notta,F.et al.Evolution of human BCR-ABL1 lymphoblastic leukaemia-initiating structural variants.A complete description of the materials and methods is pro- cells.Nature469.362-367(20111. vided in Supplementary Information.All sequence variants for the AML tumour 22. Ding,L.et al.Genome remodelling in a basal-like breast cancer metastasis and xenograft.Nature 464,999-1005(2010) samples from eight cases have been submitted to dbGaP (accession number 23. Shah,S.P.et al.Mutational evolution in a lobular breast tumour profiled at single phs000I59.v4.p2). nucleotide resolution.Nature 461,809-813(2009). 24.Yachida,S.et al Distant metastasis ccurs late during the genetic evolution of Received 29 March;accepted 29 November 2011 pancreatic cancer.Nature 467,1114-1117 (2010). Published online 11 January 2012. Navin,N.et al.Tumour evolution inferred by single-cell sequencing.Nature 472, 90-942011). 1.Testa,J.R.,Mintz,U.,Rowley,J.D.Vardiman,J.W.Golomb,H.M.Evolution of 26.Vago,L.etal Loss of mism natched HLA in leukemia after stem-cell transplantation karyotypes in acute nonlymphocytic leukemia.Cancer Res.39,3619-3627 N.Engl.1Med.361,478-488(2009) (1979) 27.Villalobos,I.B.et al.Relapse of leukemia with loss of mismatched HLA resulting 2 Garson,O.M.et al.Cytogenetic studies of 103 patients with acute myelogenous from uniparental disomy after haploidentical hematopoietic stem cell transplantation.Blood 115,3158-3161 (2010). leukemia in relapse.Cancer Genet Cytogenet 40,187-202(1989). 38 3. Ley,T.J.et al.DNA sequencing of a cytogenetically normal acute myeloid Larson,D.E.et al.SomaticSniper:identification of somatic point mutations in leukaemia genome.Nature 456,66-72 (2008). whole genome sequencing data.Bioinformatics (in the press). 4. Li,H.et al.The Sequence Alignment/Map format and SAMtools.Bioinformatics 25, Supplementary Information is linked to the online version of the paper at 2078-2079(2009). www.nature.com/nature. 5. Chen,K.et al BreakDancer:an algorithm for high-resolution mapping of genomic structural variation.Nature Methods 6,677-681 (2009). Acknowledgements We thank the Analysis Pipeline group for developing the 6. Koboldt,D.C.et al.VarScan:variant detection in massively parallel sequencingo automated sequence analysis pipelines;the LIMS group for developing tools and individual and pooled samples.Bioinformatics 25,2283-2285(2009). software to manage samples and sequencing:the Systems group for providing the IT 7. Mardis,ER.et al Recurring mutations found by sequencing an acute myeloid infrastructure and HPC solutions required for sequencing and analysis;and leukemia genome.N.Engl.J.Med.361,1058-1066(2009). R.T.Demeter for experimental support We also thank The Cancer Genome Atlas for 8. Ley,T.J.etal.DNMT3A mutations in acute myeloid leukemia.N.Engl.J.Med.363, allowing us to use unpublished data for this study,and the Washington University 2424-2433(2010). Cancer Genome Initiative for their support.This work was funded by grants to R.K.W. 9. Nakao,M.etal Inte of the flt3 gene found in acute myeloid and the National Human Genome Research Institute(NHGRI U54 HG003079),and leukemia.Leukemia 10,1911-1918(1996). grants to T.J.L from the National Cancer Institute(PO1 CA101937)and the 10.Falini,B.etal.Cytoplasmic nucleophosmin in acute myelogenous leukemia with a Barnes-Jewish Hospital Foundation(00335-0505-02). normal karyotype.N.Engl.J.Med.352,254-266(2005). Author Contributions TJ.L,L.D..J.F.D.ER.M.and R.K.W.designed the experiments. 11.Ward,P.S.et al.The common feature of leukemia-associated IDH1 and IDH2 LD.and TJ.L.led dataanalysis.LD.D.EL.CA.M..D.C.K..J.S.W.M.D.M..J.W.W.C.L..D.S. mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.Cancer Cell 17,225-234 (2010). C.C.H.K.C.H.S.,J.K.-V,M.C.W.M.AW.W.D.S.,J.E.P.and S.K.performed data analysis J.F.M.,M.D.M.and L.D.prepared figuresandtables.J.S.W..J.K.R.,M.AY.,T.L.R.S.F.LLF. 12.King-Underwood,L,Renshaw,J.Pritchard-Jones,K.Mutations in the Wilms VJ.M.,LS.,LC.S.D.M.and T.L.V.performed laboratory experiments.S.H.and P.W. tumor gene WT1 in leukemias.Blood 87,2171-2179(1996). provided samples and clinical data.DJ.D.provided informatics support TJ.L.D.C.L. 13.Gao,J.et al.Isolation of a yeast artificial chromosome spanning the 8:21 M.H.T.E.R.M.R.K.W.and J.F.D.developed projectconcept.LD..TJ.L.MJ.W.TA.G.and translocation breakpoint t(8:21)(q22:q22.3)in acute myelogenous leukemia. Proc.Natl Acad.Sci USA 88.4882-4886 (1991) J.F.D.wrote the manuscript. 14.Kirito,K.etal A novel RUNX1 mutation in familial platelet disorder with propensity Author Information All sequence variants for the AML tumour samples from eight to develop myeloid malignancies.Haematologica 93,155-156(2008). cases have been submitted to dbGaP under ac ession number phs000159.v4.p2. 15.Van Vlierberghe,P.et al.PHF6 mutations in adult acute myeloid leukemia. Reprints and permissions information is available at www.nature.com/reprints.This Leukemia25,130-134(2011). paper is distributed under the terms of the Creative Commons 16.Barjesteh van Waalwijk van Doorn-Khosrovani,S.et al.Somatic heterozygous Attribution-Non-Commercial-Share Alike licence,and is freely available to all readers at mutations in ETV6 (TEL)and frequent absence of ETV6 protein in acute myeloid www.nature.com/nature.The authors declare no competing financial interests. 1 eukemia..0 ncogene24,4129-4137(2005). Readers are welcome to comment on the online version of this article at 17.Puente.X.S.et al.Whole-genome sequencing identifies recurrent mutations in www.nature.com/nature.Correspondence and requests for materials should be chronic lymphocytic leukaemia.Nature 475,101-105(2011). addressed to T_J.L (timley@wustledu). 510 I NATURE VOL 481 26 JANUARY 2012 2012 Macmillan Publishers Limited.All rights reservedvariations were identified using BreakDancer5 . All predicted non-repetitive so￾matic SNVs, indels and all structural variants were included on custom sequence capture arrays from Roche Nimblegen. Illumina 2 3 100-bp paired-end sequen￾cing reads were produced after elution from capture arrays. VarScan6 and a read remapping strategy using Crossmatch (P. Green, unpublished data) and BWA were used for determining the validation status of predicted SNVs, indels and structural variants. A complete description of the materials and methods is pro￾vided in Supplementary Information. All sequence variants for the AML tumour samples from eight cases have been submitted to dbGaP (accession number phs000159.v4.p2). Received 29 March; accepted 29 November 2011. Published online 11 January 2012. 1. Testa, J. R., Mintz, U., Rowley, J. D., Vardiman, J. W. & Golomb, H. M. Evolution of karyotypes in acute nonlymphocytic leukemia. Cancer Res. 39, 3619–3627 (1979). 2. Garson, O. M. et al. Cytogenetic studies of 103 patients with acute myelogenous leukemia in relapse. Cancer Genet. Cytogenet. 40, 187–202 (1989). 3. Ley, T. J. et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66–72 (2008). 4. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). 5. Chen, K. et al. BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nature Methods 6, 677–681 (2009). 6. Koboldt, D. C. et al. VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics 25, 2283–2285 (2009). 7. Mardis, E. R. et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N. Engl. J. Med. 361, 1058–1066 (2009). 8. Ley, T. J. et al. DNMT3A mutations in acute myeloid leukemia. N. Engl. J. Med. 363, 2424–2433 (2010). 9. Nakao, M. et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 10, 1911–1918 (1996). 10. Falini, B. et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N. Engl. J. Med. 352, 254–266 (2005). 11. Ward, P. S. et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 17, 225–234 (2010). 12. King-Underwood, L., Renshaw, J. & Pritchard-Jones, K. Mutations in the Wilms’ tumor gene WT1 in leukemias. Blood 87, 2171–2179 (1996). 13. Gao, J. et al. Isolation of a yeast artificial chromosome spanning the 8;21 translocation breakpoint t(8;21)(q22;q22.3) in acute myelogenous leukemia. Proc. Natl Acad. Sci. USA 88, 4882–4886 (1991). 14. Kirito, K. et al. A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies. Haematologica 93, 155–156 (2008). 15. Van Vlierberghe, P. et al. PHF6 mutations in adult acute myeloid leukemia. Leukemia 25, 130–134 (2011). 16. Barjesteh van Waalwijk van Doorn-Khosrovani, S. et al. Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia. Oncogene 24, 4129–4137 (2005). 17. Puente, X. S. et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature 475, 101–105 (2011). 18. Ding, L. et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455, 1069–1075 (2008). 19. Mullighan, C. G. et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 322, 1377–1380 (2008). 20. Anderson, K. et al. Genetic variegation of clonal architecture and propagating cells in leukaemia. Nature 469, 356–361 (2011). 21. Notta, F. et al. Evolution of human BCR-ABL1 lymphoblastic leukaemia-initiating cells. Nature 469, 362–367 (2011). 22. Ding, L. et al. Genome remodelling in a basal-like breast cancer metastasis and xenograft. Nature 464, 999–1005 (2010). 23. Shah, S. P. et al. Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature 461, 809–813 (2009). 24. Yachida, S. et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 467, 1114–1117 (2010). 25. Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90–94 (2011). 26. Vago, L. et al. Loss of mismatched HLA in leukemia after stem-cell transplantation. N. Engl. J. Med. 361, 478–488 (2009). 27. Villalobos, I. B. et al. Relapse of leukemia with loss of mismatched HLA resulting from uniparental disomy after haploidentical hematopoietic stem cell transplantation. Blood 115, 3158–3161 (2010). 28. Larson, D. E. et al. SomaticSniper: identification of somatic point mutations in whole genome sequencing data. Bioinformatics (in the press). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank the Analysis Pipeline group for developing the automated sequence analysis pipelines; the LIMS group for developing tools and software to manage samples and sequencing; the Systems group for providing the IT infrastructure and HPC solutions required for sequencing and analysis; and R. T. Demeter for experimental support. We also thank The Cancer Genome Atlas for allowing us to use unpublished data for this study, and the Washington University Cancer Genome Initiative for their support. This work was funded by grants to R.K.W. and the National Human Genome Research Institute (NHGRI U54 HG003079), and grants to T.J.L. from the National Cancer Institute (PO1 CA101937) and the Barnes-Jewish Hospital Foundation (00335-0505-02). Author Contributions T.J.L., L.D., J.F.D., E.R.M. and R.K.W. designed the experiments. L.D. and T.J.L. led data analysis. L.D., D.E.L., C.A.M., D.C.K., J.S.W., M.D.M., J.W.W., C.L., D.S., C.C.H., K.C., H.S., J.K.-V., M.C.W., M.A.W., W.D.S., J.E.P. and S.K. performed data analysis. J.F.M.,M.D.M. and L.D. prepared figures and tables. J.S.W., J.K.R., M.A.Y., T.L., R.S.F., L.L.F., V.J.M., L.S., L.C., S.D.M. and T.L.V. performed laboratory experiments. S.H. and P.W. provided samples and clinical data. D.J.D. provided informatics support. T.J.L., D.C.L., M.H.T., E.R.M., R.K.W. and J.F.D. developed project concept. L.D., T.J.L., M.J.W., T.A.G. and J.F.D. wrote the manuscript. Author Information All sequence variants for the AML tumour samples from eight cases have been submitted to dbGaP under accession number phs000159.v4.p2. Reprints and permissions information is available at www.nature.com/reprints. This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence, and is freely available to all readers at www.nature.com/nature. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. Correspondence and requests for materials should be addressed to T.J.L. (timley@wustl.edu). RESEARCH LETTER 510 | NATURE | VOL 481 | 26 JANUARY 2012 ©2012 Macmillan Publishers Limited. All rights reserved