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Assembly of long, error-prone reads using repeat graphs

Nature Biotechnology volume 37, pages 540–546 (2019)Cite this article

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Abstract

Accurate genome assembly is hampered by repetitive regions. Although long single molecule sequencing reads are better able to resolve genomic repeats than short-read data, most long-read assembly algorithms do not provide the repeat characterization necessary for producing optimal assemblies. Here, we present Flye, a long-read assembly algorithm that generates arbitrary paths in an unknown repeat graph, called disjointigs, and constructs an accurate repeat graph from these error-riddled disjointigs. We benchmark Flye against five state-of-the-art assemblers and show that it generates better or comparable assemblies, while being an order of magnitude faster. Flye nearly doubled the contiguity of the human genome assembly (as measured by the NGA50 assembly quality metric) compared with existing assemblers.

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Fig. 1: Flye outline.
Fig. 2: Constructing the approximate repeat graph from local self-alignments.
Fig. 3: Resolving an unbridged repeat.
Fig. 4: An SD from the Flye assembly of the HUMAN dataset and the distribution of the lengths and complexities of all SDs from the same assembly.
Fig. 5: Constructing the repeat plot of a tour in the graph and constructing the repeat graph from a repeat plot.

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Data availability

All described datasets are publicly available through the corresponding repositories. The supplementary files, including the assemblies generated by Flye, are available at https://doi.org/10.5281/zenodo.1143753; NCTC PacBio reads: http://www.sanger.ac.uk/resources/downloads/bacteria/nctc/; PacBio metagenome dataset: https://github.com/PacificBiosciences/DevNet/wiki/Human_Microbiome_Project_MockB_Shotgun; PacBio C. elegans dataset: https://github.com/PacificBiosciences/DevNet/wiki/C.-elegans-data-set; PacBio/ONT S. cerevisiae dataset: https://github.com/fg6/YeastStrainsStudy. The ONT reads from the HUMAN and HUMAN+ datasets are available at https://github.com/nanopore-wgs-consortium/NA12878. The matching Illumina reads are available as SRA project ERP001229. The Canu HUMAN+ assembly was downloaded from https://genomeinformatics.github.io/na12878update. MaSuRCA assemblies are available from http://masurca.blogspot.com/.

Code availability

The Flye code used in this study is available in the online version of the paper. The most recent Flye version is freely available at http://github.com/fenderglass/Flye.

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Acknowledgements

We are indebted to S. Nurk for his multiple rounds of critique and suggestions that have improved the paper. We are also grateful to A. Mikheenko, B. Behsaz, L. Pu, and G. Tesler for their comments. This work is supported by NSF/MCB-BSF grant no. 1715911.

Author information

Authors and Affiliations

  1. Department of Computer Science and Engineering, University of California, San Diego, CA, USA

    Mikhail Kolmogorov & Pavel A. Pevzner

  2. Graduate Program in Bioinformatics and Systems Biology, University of California, San Diego, CA, USA

    Jeffrey Yuan

  3. Research School of Computer Science, Australian National University, Canberra, Australian Capital Territory, Australia

    Yu Lin

Authors
  1. Mikhail Kolmogorov
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  2. Jeffrey Yuan
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  4. Pavel A. Pevzner
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Contributions

All authors contributed to developing the Flye algorithms and writing the paper. M.K., Y.L., and J.Y. implemented the Flye algorithm. M.K. benchmarked Flye and other assembly tools. P.A.P. directed the work.

Corresponding author

Correspondence to Pavel A. Pevzner.

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Integrated supplementary information

Supplementary Figure 1 A comparison of the Flye and HINGE assembly graphs on bacterial genomes from the BACTERIA dataset.

(Left) The Flye and Hinge assembly graphs of the KP9657 dataset. There is a single unique edge entering into (and exiting) the unresolved “yellow” repeat and connecting it to the rest of the graph. Thus, this repeat can be resolved if one excludes the possibility that it is shared between a chromosome and a plasmid. In contrast to HINGE, Flye does not rule out this possibility and classifies the yellow repeat as unresolved. (Right) The Flye and Hinge assembly graphs of the EC10864 dataset show a mosaic repeat of multiplicity four formed by yellow, blue, red and green edges (the two copies of each edge represent complementary strands). HINGE reports a complete assembly into a single chromosome.

Supplementary Figure 2 The assembly graph of the YEAST-ONT dataset.

Edges that were classified as repetitive by Flye are shown in color, while unique edges are black. Flye assembled the YEAST-ONT dataset into a graph with 21 unique and 34 repeat edges and generated 21 contigs as unambiguous paths in the assembly graph. A path v1, …vi, vi+1 …vn in the graph is called unambiguous if there exists a single incoming edge into each vertex of this path before vi+1 and a single outgoing edge from each vertex after vi. Each unique contig is formed by a single unique edge and possibly multiple repeat edges, while repetitive contigs consist of the repetitive edges which were not covered by the unique contigs. The visualization was generated using the graphviz tool (http://graphviz.org).

Supplementary Figure 3 The assembly graph of the WORM dataset.

Edges that were classified as repetitive by Flye are shown in color, while unique edges are black. Flye assembled the WORM dataset into a graph with 127 unique and 61 repeat edges and generated 127 contigs as unambiguous paths in the assembly graph. The visualization was generated using the graphviz tool (http://graphviz.org).

Supplementary Figure 4 Dot plots showing the alignment of reads against the Flye assembly, the Miniasm assembly and the reference C. elegans genome.

(a) The reference genome contains a tandem repeat of length 1.9 kb (10 copies) on chromosome X with the repeated unit having length ≈190 nucleotides. In contrast, the Flye and Miniasm assemblies of this region suggest a tandem repeat of length 5.5 kb (27 copies) and 2.8 kb (13 copies), respectively. 15 reads that span over the tandem repeat support the Flye assembly (the mean length between the flanking unique sequence matches the repeat length reconstructed by Flye) and suggests that the Flye length estimate is more accurate. (b) The reference genome contains a tandem repeat of length 2 kb on chromosome 1. In contrast, the Flye and Miniasm assemblies of this region suggest a tandem repeat of length 10 kb and 5.6 kb, respectively. A single read that spans over the tandem repeat supports the Flye assembly. Since the mean read length in the WORM dataset is 11 kb, it is expected to have a single read spanning a given 10.0 kb region but many more reads spanning any 5.6 kb region (as implied by the Miniasm assembly) or 2.0 kb region (as implied by the reference genome). Six out of 23 reads cross the “left” border (two out of 18 reads cross the “right” border) of this tandem repeat by more than 5.6 kb, thus contradicting the length estimate given by Miniasm and suggesting that the Flye length estimate is more accurate. (c) The reference genome contains a tandem repeat of length 3 kb on chromosome X. In contrast, the Flye and Miniasm assemblies of this region suggest a tandem repeat of lengths 13.6 kb and 8 kb, respectively. A single read that spans over the tandem repeat reveals the repeat cluster to be of length 12.2k, which suggests that the Flye length estimate is more accurate. (d) The reference genome contains a tandem repeat of length 1.5 kb on chromosome 1. In contrast, the Flye and Miniasm assemblies of this region suggest tandem repeats of length 17 kb and 4.3 kb, respectively. One read that spans over the tandem repeat reveals the repeat cluster to be of length 18.0 kb, which suggests that the Flye length estimate is more accurate.

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Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Notes 1–16

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Kolmogorov, M., Yuan, J., Lin, Y. et al. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37, 540–546 (2019). https://doi.org/10.1038/s41587-019-0072-8

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