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Scalable telomere-to-telomere assembly for diploid and polyploid genomes with double graph

Nature Methods (2024)Cite this article

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Abstract

Despite advances in long-read sequencing technologies, constructing a near telomere-to-telomere assembly is still computationally demanding. Here we present hifiasm (UL), an efficient de novo assembly algorithm combining multiple sequencing technologies to scale up population-wide near telomere-to-telomere assemblies. Applied to 22 human and two plant genomes, our algorithm produces better diploid assemblies at a cost of an order of magnitude lower than existing methods, and it also works with polyploid genomes.

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Fig. 1: Hybrid assembly with PacBio HiFi and ONT ultra-long reads.
Fig. 2: Statistics of different assemblies.

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Data availability

For the human reference genome, GRCh38 and CHM13v2; HiFi reads of HPRC year 2 samples except HG002: https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=submissions/1E2DD570-3B26-418B-B50F-5417F64C5679--HIFI_DEEPCONSENSUS/; ONT ultra-long reads of HPRC year 2 samples except HG002 (R9.4.1 flow cells): https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=submissions/90A1F283-2752-438B-917F-53AE76C9C43E--UCSC_HPRC_nanopore_Year2/; Hi-C reads of HPRC year 2 samples except HG002: https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=submissions/4C696EB9-9AD2-47A2-8011-2F43977CC4E0--Y2-HIC/; parental short reads of HPRC year 2 samples except HG002: https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=submissions/AD30A684-C7A8-4D24-89B2-040DFF021B0C--Y2_1000G_DATA/; HG002 HiFi reads (Google Cloud Storage): https://s3-us-west-2.amazonaws.com/human-pangenomics/NHGRI_UCSC_panel/HG002/hpp_HG002_NA24385_son_v1/PacBio_HiFi/20kb/m64011_190830_220126.Q20.fastq, https://s3-us-west-2.amazonaws.com/human-pangenomics/NHGRI_UCSC_panel/HG002/hpp_HG002_NA24385_son_v1/PacBio_HiFi/20kb/m64011_190901_095311.Q20.fastq, https://s3-us-west-2.amazonaws.com/human-pangenomics/NHGRI_UCSC_panel/HG002/hpp_HG002_NA24385_son_v1/PacBio_HiFi/15kb/m64012_190920_173625.Q20.fastq, https://s3-us-west-2.amazonaws.com/human-pangenomics/NHGRI_UCSC_panel/HG002/hpp_HG002_NA24385_son_v1/PacBio_HiFi/15kb/m64012_190921_234837.Q20.fastq; HG002 ultra-long reads (R9.4.1 flow cells, pass reads only): https://s3-us-west-2.amazonaws.com/human-pangenomics/NHGRI_UCSC_panel/HG002/nanopore/ultra-long/03_08_22_R941_HG002_1_Guppy_6.1.2_5mc_cg_prom_sup.tar, https://s3-us-west-2.amazonaws.com/human-pangenomics/NHGRI_UCSC_panel/HG002/nanopore/ultra-long/03_08_22_R941_HG002_2_Guppy_6.1.2_5mc_cg_prom_sup.tar, https://s3-us-west-2.amazonaws.com/human-pangenomics/NHGRI_UCSC_panel/HG002/nanopore/ultra-long/03_08_22_R941_HG002_3_Guppy_6.1.2_5mc_cg_prom_sup.tar; HG002 parental short reads: https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=working/HPRC_PLUS/HG002/raw_data/Illumina/parents/ and HG002 Hi-C reads: ‘HG002.HiC_1*’ from https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=working/HPRC_PLUS/HG002/raw_data/hic/downsampled/. All reads of HPRC year 1 samples (R9.4.1 flow cells for ONT reads): https://github.com/human-pangenomics/HPP_Year1_Data_Freeze_v1.0. All reads of Arabidopsis (R9.4.1 flow cells for ONT reads): https://ngdc.cncb.ac.cn/search/?dbId=gsa&q=CRA004538. All reads of potato (R9.4.1 flow cells for ONT ultra-long reads): https://ngdc.cncb.ac.cn/gsa/browse/CRA006012. Hifiasm (UL) assemblies of HPRC year 2 samples: ‘*hifiasm_v0.19.5*’ from https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=submissions/53FEE631-4264-4627-8FB6-09D7364F4D3B--ASM-COMP/. Verkko assemblies of HPRC year 2 samples: ‘*verkko_1.3.1*’ from https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=submissions/53FEE631-4264-4627-8FB6-09D7364F4D3B--ASM-COMP/. All evaluated HPRC year 1 and plant assemblies are available at https://zenodo.org/record/7996422 (ref. 25) and https://zenodo.org/record/7962930 (ref. 26), respectively.

Code availability

Hifiasm (UL) along with its source code is freely available at https://github.com/chhylp123/hifiasm.

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Acknowledgements

This study was supported by US National Institutes of Health (grant nos. R01HG010040, U01HG010971 and U41HG010972 to H.L. and K99HG012798 to H.C.). We thank the HPRC for making year 1 and year 2 datasets publicly available.

Author information

Authors and Affiliations

  1. Department of Data Science, Dana-Farber Cancer Institute, Boston, MA, USA

    Haoyu Cheng & Heng Li

  2. Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA

    Haoyu Cheng & Heng Li

  3. Genomics Institute, University of California, Santa Cruz, CA, USA

    Mobin Asri & Julian Lucas

  4. Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

    Sergey Koren

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  1. Haoyu Cheng
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Contributions

H.C. and H.L. designed the algorithm, implemented hifiasm (UL) and drafted the paper. H.C. benchmarked hifiasm (UL) and other assemblers. M.A., J.L. and S.K. designed the evaluation of human genome assemblies.

Corresponding author

Correspondence to Heng Li.

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The authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks Derek Bickhart and Antoine Limasset for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Lin Tang, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Accurate HiFi string graph combining PacBio HiFi and ONT ultra-long reads.

(a) Effect of contained reads in the string graph. Rectangles in orange and blue represent heterozygous HiFi reads from haplotype 1 and haplotype 2, respectively. Green rectangles are HiFi reads originating from homozygous regions, whereas red rectangles are contained reads. The string graph is constructed using all reads, except for two contained reads. (b) Hifiasm (UL) aligns ultra-long reads to the HiFi string graph with contained reads to alleviate the contained read problem. The alignment paths of ultra-long reads from haplotype 1 and haplotype 2 are represented by orange and blue lines, respectively. Despite being a contained read, h12 is retained as the critical read because it is covered by ultra-long reads u6 and u7. To ensure accurate graph cleaning, hifiasm (UL) also tracks the number of ultra-long reads that support each edge as its weight. For instance, the edge weight between h5 and h8 is 2 because ultra-long reads u4 and u5 cover it.

Supplementary information

Supplementary Information

Supplementary Tables 1–5, Fig. 1 and text.

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Peer Review File

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Cheng, H., Asri, M., Lucas, J. et al. Scalable telomere-to-telomere assembly for diploid and polyploid genomes with double graph. Nat Methods (2024). https://doi.org/10.1038/s41592-024-02269-8

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