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De novo assembly and genotyping of variants using colored de Bruijn graphs

Abstract

Detecting genetic variants that are highly divergent from a reference sequence remains a major challenge in genome sequencing. We introduce de novo assembly algorithms using colored de Bruijn graphs for detecting and genotyping simple and complex genetic variants in an individual or population. We provide an efficient software implementation, Cortex, the first de novo assembler capable of assembling multiple eukaryotic genomes simultaneously. Four applications of Cortex are presented. First, we detect and validate both simple and complex structural variations in a high-coverage human genome. Second, we identify more than 3 Mb of sequence absent from the human reference genome, in pooled low-coverage population sequence data from the 1000 Genomes Project. Third, we show how population information from ten chimpanzees enables accurate variant calls without a reference sequence. Last, we estimate classical human leukocyte antigen (HLA) genotypes at HLA-B, the most variable gene in the human genome.

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Figure 1: Schematic representation of four methods of variation analysis using colored de Bruijn graphs; line width represents coverage.
Figure 2: Simulation-based evaluation of Cortex.
Figure 3: Structural and complex variants identified in a single high-coverage genome.
Figure 4: Population analysis with Cortex.
Figure 5: HLA-B genotyping from HTS data using Cortex.

References

  1. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  2. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  3. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).

    CAS  Article  Google Scholar 

  4. Li, R., Li, Y., Kristiansen, K. & Wang, J. SOAP: short oligonucleotide alignment program. Bioinformatics 24, 713–714 (2008).

    CAS  Article  Google Scholar 

  5. Lunter, G. & Goodson, M. Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 21, 936–939 (2011).

    CAS  Article  Google Scholar 

  6. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    CAS  Article  Google Scholar 

  7. Albers, C.A. et al. Dindel: accurate indel calls from short-read data. Genome Res. 21, 961–973 (2011).

    CAS  Article  Google Scholar 

  8. Lee, S., Hormozdiari, F., Alkan, C. & Brudno, M. MoDIL: detecting small indels from clone-end sequencing with mixtures of distributions. Nat. Methods 6, 473–474 (2009).

    CAS  Article  Google Scholar 

  9. Hajirasouliha, I. et al. Detection and characterization of novel sequence insertions using paired-end next-generation sequencing. Bioinformatics 26, 1277–1283 (2010).

    CAS  Article  Google Scholar 

  10. Handsaker, R.E., Korn, J.M., Nemesh, J. & McCarroll, S.A. Discovery and genotyping of genome structural polymorphism by sequencing on a population scale. Nat. Genet. 43, 269–276 (2011).

    CAS  Article  Google Scholar 

  11. Korbel, J.O. et al. PEMer: a computational framework with simulation-based error models for inferring genomic structural variants from massive paired-end sequencing data. Genome Biol. 10, R23 (2009).

    Article  Google Scholar 

  12. Korbel, J.O. et al. Paired-end mapping reveals extensive structural variation in the human genome. Science 318, 420–426 (2007).

    CAS  Article  Google Scholar 

  13. Mills, R.E. et al. Mapping copy number variation by population-scale genome sequencing. Nature 470, 59–65 (2011).

    CAS  Article  Google Scholar 

  14. Tuzun, E. et al. Fine-scale structural variation of the human genome. Nat. Genet. 37, 727–732 (2005).

    CAS  Article  Google Scholar 

  15. Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

    CAS  Article  Google Scholar 

  16. Wang, J. et al. The diploid genome sequence of an Asian individual. Nature 456, 60–65 (2008).

    CAS  Article  Google Scholar 

  17. 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).

  18. Ge, F., Wang, L.S. & Kim, J. The cobweb of life revealed by genome-scale estimates of horizontal gene transfer. PLoS Biol. 3, e316 (2005).

    Article  Google Scholar 

  19. Beiko, R.G., Harlow, T.J. & Ragan, M.A. Highways of gene sharing in prokaryotes. Proc. Natl. Acad. Sci. USA 102, 14332–14337 (2005).

    CAS  Article  Google Scholar 

  20. Holcomb, C.L. et al. A multi-site study using high-resolution HLA genotyping by next generation sequencing. Tissue Antigens 77, 206–217 (2011).

    CAS  Article  Google Scholar 

  21. Fonseca, V.G. et al. Second-generation environmental sequencing unmasks marine metazoan biodiversity. Nat. Commun. 1, 98 (2010).

    Article  Google Scholar 

  22. Iafrate, A.J. et al. Detection of large-scale variation in the human genome. Nat. Genet. 36, 949–951 (2004).

    CAS  Article  Google Scholar 

  23. Redon, R. et al. Global variation in copy number in the human genome. Nature 444, 444–454 (2006).

    CAS  Article  Google Scholar 

  24. Sebat, J. et al. Large-scale copy number polymorphism in the human genome. Science 305, 525–528 (2004).

    CAS  Article  Google Scholar 

  25. Sharp, A.J. et al. Segmental duplications and copy-number variation in the human genome. Am. J. Hum. Genet. 77, 78–88 (2005).

    CAS  Article  Google Scholar 

  26. Kidd, J.M. et al. Mapping and sequencing of structural variation from eight human genomes. Nature 453, 56–64 (2008).

    CAS  Article  Google Scholar 

  27. Myers, E.W. Toward simplifying and accurately formulating fragment assembly. J. Comput. Biol. 2, 275–290 (1995).

    CAS  Article  Google Scholar 

  28. Myers, E.W. The fragment assembly string graph. Bioinformatics 21 (suppl. 2), ii79–ii85 (2005).

    CAS  PubMed  Google Scholar 

  29. Simpson, J.T. & Durbin, R. Efficient construction of an assembly string graph using the FM-index. Bioinformatics 26, i367–i373 (2010).

    CAS  Article  Google Scholar 

  30. Zerbino, D.R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008).

    CAS  Article  Google Scholar 

  31. Gnerre, S. et al. High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc. Natl. Acad. Sci. USA 108, 1513–1518 (2011).

    CAS  Article  Google Scholar 

  32. Li, R. et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 20, 265–272 (2010).

    CAS  Article  Google Scholar 

  33. Jones, T. et al. The diploid genome sequence of Candida albicans. Proc. Natl. Acad. Sci. USA 101, 7329–7334 (2004).

    CAS  Article  Google Scholar 

  34. Vinson, J.P. et al. Assembly of polymorphic genomes: algorithms and application to Ciona savignyi. Genome Res. 15, 1127–1135 (2005).

    Article  Google Scholar 

  35. Kim, J.H., Waterman, M.S. & Li, L.M. Diploid genome reconstruction of Ciona intestinalis and comparative analysis with Ciona savignyi. Genome Res. 17, 1101–1110 (2007).

    CAS  Article  Google Scholar 

  36. Donmez, N. & Brudno, M. Hapsembler: an assembler for highly polymorphic genomes. in Research in Computational Molecular Biology, Lecture Notes in Computer Science Vol. 6577 (eds. Bafna, V. & Sahinalp, S.), 38–52 (Springer, Berlin, Heidelberg, 2011).

  37. Pevzner, P.A., Tang, H. & Waterman, M.S. An Eulerian path approach to DNA fragment assembly. Proc. Natl. Acad. Sci. USA 98, 9748–9753 (2001).

    CAS  Article  Google Scholar 

  38. Idury, R.M. & Waterman, M.S. A new algorithm for DNA sequence assembly. J. Comput. Biol. 2, 291–306 (1995).

    CAS  Article  Google Scholar 

  39. Simpson, J.T. et al. ABySS: a parallel assembler for short read sequence data. Genome Res. 19, 1117–1123 (2009).

    CAS  Article  Google Scholar 

  40. Zerbino, D.R., McEwen, G.K., Margulies, E.H. & Birney, E. Pebble and rock band: heuristic resolution of repeats and scaffolding in the velvet short-read de novo assembler. PLoS ONE 4, e8407 (2009).

    Article  Google Scholar 

  41. Kidd, J.M. et al. A human genome structural variation sequencing resource reveals insights into mutational mechanisms. Cell 143, 837–847 (2010).

    CAS  Article  Google Scholar 

  42. Myers, S. et al. Drive against hotspot motifs in primates implicates the PRDM9 gene in meiotic recombination. Science 327, 876–879 (2010).

    CAS  Google Scholar 

  43. The International HapMap Consortium. et al. A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851–861 (2007).

  44. de Bakker, P.I. et al. A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC. Nat. Genet. 38, 1166–1172 (2006).

    CAS  Article  Google Scholar 

  45. Ratan, A., Yu, Z., Hayes, V.M., Schuster, S.C. & Miller, W. Calling SNPs without a reference sequence. BMC Bioinformatics 11, 130 (2010).

    Article  Google Scholar 

  46. Peterlongo, P., Schnel, N., Pisanti, N., Sagot, M.-F. & Lacroix, V. Identifying SNPs without a reference genome by comparing raw reads. in String Processing and Information Retrieval—17th International Symposium (eds. Chavez, E. & Lonardi, S.) 147–158 (Los Cabos, Mexico, 2010).

  47. Ding, L., Wendl, M.C., Koboldt, D.C. & Mardis, E.R. Analysis of next-generation genomic data in cancer: accomplishments and challenges. Hum. Mol. Genet. 19, R188–R196 (2010).

    CAS  Article  Google Scholar 

  48. Harris, S.R. et al. Evolution of MRSA during hospital transmission and intercontinental spread. Science 327, 469–474 (2010).

    CAS  Article  Google Scholar 

  49. Butler, J. et al. ALLPATHS: de novo assembly of whole-genome shotgun microreads. Genome Res. 18, 810–820 (2008).

    CAS  Article  Google Scholar 

  50. Chaisson, M.J., Brinza, D. & Pevzner, P.A. De novo fragment assembly with short mate-paired reads: does the read length matter? Genome Res. 19, 336–346 (2009).

    CAS  Article  Google Scholar 

  51. Kelley, D.R., Schatz, M.C. & Salzberg, S.L. Quake: quality-aware detection and correction of sequencing errors. Genome Biol. 11, R116 (2010).

    CAS  Article  Google Scholar 

  52. Allsopp, C.E. et al. Sequence analysis of HLA-Bw53, a common West African allele, suggests an origin by gene conversion of HLA-B35. Hum. Immunol. 30, 105–109 (1991).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We would like to thank the members of the 1000 Genomes Project Consortium for discussion, suggestions and sequencing data. We thank B. Ahiska, A. Auton, E. Birney, R. Durbin, G. Lunter, J. Woolf and D. Zerbino for discussion, two anonymous reviewers for their comments and members of the PanMap Project and the Genomics Core at the Wellcome Trust Centre for Human Genetics for access to sequence data. Z.I. is funded by a grant from the Wellcome Trust (WT086084/Z/08/Z to G.M.). The sequencing of NA12878 was performed by the Wellcome Trust Sequencing Core at Oxford, under a grant from the Wellcome Trust (090532/Z/09/Z).

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Contributions

Z.I. and G.M. designed the study, developed the mathematical models and wrote the manuscript. M.C. and Z.I. developed the variant discovery algorithms, designed the multicolor graph data structures and implemented software. Z.I. performed simulations and analyses for cases 1, 3 and 4. I.T. and Z.I. performed analyses for case 2. P.F. contributed to early plans for Cortex.

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Correspondence to Gil McVean.

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Supplementary Note, Supplementary Figures 1–6 and Supplementary Tables 1–7 (PDF 1207 kb)

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Iqbal, Z., Caccamo, M., Turner, I. et al. De novo assembly and genotyping of variants using colored de Bruijn graphs. Nat Genet 44, 226–232 (2012). https://doi.org/10.1038/ng.1028

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