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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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 (https://hpc.nih.gov).

Author information

Author notes

    • Sergey Koren
    •  & Arang Rhie

    These authors contributed equally to this work.


  1. Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland, USA.

    • Sergey Koren
    • , Arang Rhie
    • , Brian P Walenz
    • , Alexander T Dilthey
    •  & Adam M Phillippy
  2. Institute of Medical Microbiology, Heinrich-Heine-University Düsseldorf, Düsseldorf, North Rhine-Westphalia, Germany.

    • Alexander T Dilthey
  3. Cell Wall Biology and Utilization Laboratory, ARS USDA, Madison, Wisconsin, USA.

    • Derek M Bickhart
  4. Pacific Biosciences, Menlo Park, California, USA.

    • Sarah B Kingan
  5. Davies Research Centre, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy SA, Australia.

    • Stefan Hiendleder
    •  & John L Williams
  6. Robinson Research Institute, The University of Adelaide, Adelaide SA, Australia.

    • Stefan Hiendleder
  7. US Meat Animal Research Center, ARS USDA, Clay Center, Nebraska, USA.

    • Timothy P L Smith


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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.

Competing interests

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

Corresponding authors

Correspondence to Timothy P L Smith or Adam M Phillippy.

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