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De novo assembly of haplotype-resolved genomes with trio binning


Complex allelic variation hampers the assembly of haplotype-resolved sequences from diploid genomes. We developed trio binning, an approach that simplifies haplotype assembly by resolving allelic variation before assembly. In contrast with prior approaches, the effectiveness of our method improved with increasing heterozygosity. Trio binning uses short reads from two parental genomes to first partition long reads from an offspring into haplotype-specific sets. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. We used trio binning to recover both haplotypes of a diploid human genome and identified complex structural variants missed by alternative approaches. We sequenced an F1 cross between the cattle subspecies Bos taurus taurus and Bos taurus indicus and completely assembled both parental haplotypes with NG50 haplotig sizes of >20 Mb and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We suggest that trio binning improves diploid genome assembly and will facilitate new studies of haplotype variation and inheritance.

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Figure 1: Outline of trio binning and haplotype assembly.
Figure 2: Effect of data characteristics on trio binning.
Figure 3: Read and assembly k-mer statistics for an Arabidopsis thaliana F1 hybrid.
Figure 4: Haplotype variation in a diploid human genome.
Figure 5: Diploid assembly of a Bos taurus F1 hybrid.

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Primary accessions



  1. Phillippy, A.M. New advances in sequence assembly. Genome Res. 27, xi–xiii (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Koren, S. et al. Reducing assembly complexity of microbial genomes with single-molecule sequencing. Genome Biol. 14, R101 (2013).

    PubMed  PubMed Central  Google Scholar 

  3. Korlach, J. et al. De novo PacBio long-read and phased avian genome assemblies correct and add to reference genes generated with intermediate and short reads. Gigascience 6, 1–16 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Myers, E.W. et al. A whole-genome assembly of Drosophila. Science 287, 2196–2204 (2000).

    CAS  PubMed  Google Scholar 

  5. Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002).

  6. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

  7. Cao, H. et al. De novo assembly of a haplotype-resolved human genome. Nat. Biotechnol. 33, 617–622 (2015).

    CAS  PubMed  Google Scholar 

  8. Steinberg, K.M. et al. Single haplotype assembly of the human genome from a hydatidiform mole. Genome Res. 24, 2066–2076 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Schneider, V.A. et al. Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly. Genome Res. 27, 849–864 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Chaisson, M.J. et al. Resolving the complexity of the human genome using single-molecule sequencing. Nature 517, 608–611 (2015).

    CAS  PubMed  Google Scholar 

  11. Batzoglou, S. et al. ARACHNE: a whole-genome shotgun assembler. Genome Res. 12, 177–189 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  13. Levy, S. et al. The diploid genome sequence of an individual human. PLoS Biol. 5, e254 (2007).

    PubMed  PubMed Central  Google Scholar 

  14. Patterson, M. et al. WhatsHap: weighted haplotype assembly for future-generation sequencing reads. J. Comput. Biol. 22, 498–509 (2015).

    CAS  PubMed  Google Scholar 

  15. Edge, P., Bafna, V. & Bansal, V. HapCUT2: robust and accurate haplotype assembly for diverse sequencing technologies. Genome Res. 27, 801–812 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Larkin, D.M. et al. Whole-genome resequencing of two elite sires for the detection of haplotypes under selection in dairy cattle. Proc. Natl. Acad. Sci. USA 109, 7693–7698 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Yang, H., Chen, X. & Wong, W.H. Completely phased genome sequencing through chromosome sorting. Proc. Natl. Acad. Sci. USA 108, 12–17 (2011).

    CAS  PubMed  Google Scholar 

  18. Falconer, E. & Lansdorp, P.M. Strand-seq: a unifying tool for studies of chromosome segregation. Semin. Cell Dev. Biol. 24, 643–652 (2013).

    CAS  PubMed  Google Scholar 

  19. Selvaraj, S., R Dixon, J., Bansal, V. & Ren, B. Whole-genome haplotype reconstruction using proximity-ligation and shotgun sequencing. Nat. Biotechnol. 31, 1111–1118 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Chin, C.S. et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat. Methods 13, 1050–1054 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Weisenfeld, N.I., Kumar, V., Shah, P., Church, D.M. & Jaffe, D.B. Direct determination of diploid genome sequences. Genome Res. 27, 757–767 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Seo, J.S. et al. De novo assembly and phasing of a Korean human genome. Nature 538, 243–247 (2016).

    CAS  PubMed  Google Scholar 

  23. Porubsky, D. et al. Dense and accurate whole-chromosome haplotyping of individual genomes. Nat. Commun. 8, 1293 (2017).

    PubMed  PubMed Central  Google Scholar 

  24. Matthews, B.J. et al. Improved Aedes aegypti mosquito reference genome assembly enables biological discovery and vector control. Nature (in the press).

  25. International HapMap Consortium. The International HapMap Project. Nature 426, 789–796 (2003).

  26. The 1000 Genomes Project Consortium. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012).

  27. Eberle, M.A. et al. A reference data set of 5.4 million phased human variants validated by genetic inheritance from sequencing a three-generation 17-member pedigree. Genome Res. 27, 157–164 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Malinsky, M., Simpson, J.T. & Durbin, R. trio-sga: facilitating de novo assemblyof highly heterozygous genomes with parent–child trios. bioRxiv Preprint at (2016).

  29. Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722–736 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Vurture, G.W. et al. GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics 33, 2202–2204 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Waterhouse, R.M. et al. BUSCO applications from quality assessments to gene prediction and phylogenomics. Mol. Biol. Evol. (2017).

    PubMed Central  Google Scholar 

  32. Salzberg, S.L. et al. GAGE: a critical evaluation of genome assemblies and assembly algorithms. Genome Res. 22, 557–567 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Kurtz, S. et al. Versatile and open software for comparing large genomes. Genome Biol. 5, R12 (2004).

    PubMed  PubMed Central  Google Scholar 

  34. Nattestad, M. & Schatz, M.C. Assemblytics: a web analytics tool for the detection of variants from an assembly. Bioinformatics 32, 3021–3023 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Lamesch, P. et al. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 40, D1202–D1210 (2012).

    CAS  PubMed  Google Scholar 

  36. Dilthey, A.T. et al. High-accuracy HLA type inference from whole-genome sequencing data using population reference graphs. PLoS Comput. Biol. 12, e1005151 (2016).

    PubMed  PubMed Central  Google Scholar 

  37. Zimin, A.V. et al. A whole-genome assembly of the domestic cow, Bos taurus. Genome Biol. 10, R42 (2009).

    PubMed  PubMed Central  Google Scholar 

  38. Canavez, F.C. et al. Genome sequence and assembly of Bos indicus. J. Hered. 103, 342–348 (2012).

    CAS  PubMed  Google Scholar 

  39. Zhou, Y. et al. Genome-wide CNV analysis reveals variants associated with growth traits in Bos indicus. BMC Genomics 17, 419 (2016).

    PubMed  PubMed Central  Google Scholar 

  40. Sedlazeck, F.J. et al. Accurate detection of complex structural variations using single-molecule sequencing. Nat. Methods 15, 461–468 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Jain, M. et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat. Biotechnol. 36, 338–345 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Li, H. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. Bioinformatics 32, 2103–2110 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Kolmogorov, M., Yuan, J., Lin, Y. & Pevzner, P. Assembly of long error-prone reads using repeat graphs. bioRxiv Preprint at (2018).

  44. Chaisson, M.J., Mukherjee, S., Kannan, S. & Eichler, E.E. Duplications de novo using polyploid phasing. in International Conference on Research in Computational Molecular Biology (ed. Sahinalp S.) 117–133 (Springer, 2017).

  45. English, A.C. et al. Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology. PLoS One 7, e47768 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Chin, C.S. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569 (2013).

    CAS  PubMed  Google Scholar 

  47. Loman, N.J., Quick, J. & Simpson, J.T. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat. Methods 12, 733–735 (2015).

    CAS  PubMed  Google Scholar 

  48. Ma, B., Tromp, J. & Li, M. PatternHunter: faster and more sensitive homology search. Bioinformatics 18, 440–445 (2002).

    CAS  PubMed  Google Scholar 

  49. Nattestad, M., Chin, C.-S. & Schatz, M.C. Ribbon: visualizing complex genome alignments and structural variation. bioRxiv Preprint at (2016).

  50. Mapleson, D., Garcia Accinelli, G., Kettleborough, G., Wright, J. & Clavijo, B.J. KAT: a K-mer analysis toolkit to quality control NGS datasets and genome assemblies. Bioinformatics 33, 574–576 (2017).

    CAS  PubMed  Google Scholar 

  51. Kajitani, R. et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 24, 1384–1395 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Fofanov, Y. et al. How independent are the appearances of n-mers in different genomes? Bioinformatics 20, 2421–2428 (2004).

    CAS  PubMed  Google Scholar 

  53. Dilthey, A., Cox, C., Iqbal, Z., Nelson, M.R. & McVean, G. Improved genome inference in the MHC using a population reference graph. Nat. Genet. 47, 682–688 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at (2013).

  55. Garrison, E. & Marth, G. Haplotype-based variant detection from short-read sequencing. Preprint at (2012).

  56. Casper, J. et al. The UCSC Genome Browser database: 2018 update. Nucleic Acids Res. 46, D762–D769 (2018).

    CAS  PubMed  Google Scholar 

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We thank W, Thompson, K. Kuhn, K. McClure and R. Lee for technical assistance, and T. Graves-Lindsay and Washington University in St. Louis for public release of the PacBio NA12878 data. S.K., A.R., B.P.W. and A.M.P. were supported by the Intramural Research Program of the National Human Genome Research Institute, US National Institutes of Health. S.H. and J.L.W. were funded from the JS Davies bequest to the University of Adelaide. T.P.L.S. was supported by USDA-ARS Project 3040-31000-100-00D. D.M.B. was supported by USDA-ARS Project 5090-31000-026-00-D. This research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C2098). This work used the computational resources of the NIH HPC Biowulf cluster (

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Authors and Affiliations



A.M.P. and T.P.L.S. conceived and coordinated the project. S.K. and A.R. designed the trio-binning method. S.K., A.R. and B.P.W. implemented the software. S.K., A.R., B.P.W., A.T.D., D.M.B., S.B.K. and A.M.P. performed analyses. S.H. designed and performed breeding experiments and sample collections. J.L.W. contributed to development of the concept and provision of samples. T.P.L.S. performed sequencing. S.K., A.R., T.P.L.S., J.L.W. and A.M.P. wrote the manuscript. All of the authors approved the final manuscript.

Corresponding authors

Correspondence to Timothy P L Smith or Adam M Phillippy.

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Competing interests

S.B.K. is a current employee of Pacific Biosciences.

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Koren, S., Rhie, A., Walenz, B. et al. De novo assembly of haplotype-resolved genomes with trio binning. Nat Biotechnol 36, 1174–1182 (2018).

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