PAG XXIV: From Sequencing to Chromosomes: New de novo Assembly and Scaffolding Methods Improve the Goat Reference Genome

Single-molecule sequencing is now routinely used to assemble complete, high-quality microbial genomes, but these assembly methods have not scaled well to large genomes. To address this problem, we previously introduced the MinHash Alignment Process (MHAP) for overlapping single-molecule reads using probabilistic, locality-sensitive hashing. Integrating MHAP with Celera Assembler (CA) has enabled reference-grade assemblies of model organisms, revealing novel heterochromatic sequences and filling low-complexity gap sequences in the GRCh38 human reference genome. We have applied our methods to assemble the San Clemente goat genome. Combining single-molecule sequencing from Pacific Biosciences and BioNano Genomics generates and assembly that is over 150-fold more contiguous than the latest Capra hircus reference. In combination with Hi-C sequencing, the assembly surpasses reference assemblies, de novo, with minimal manual intervention. The autosomes are each assembled into a single scaffold. Our assembly provides a more complete gene reconstruction, better alignments with Goat 52k chip, and improved allosome reconstruction. In addition to providing increased continuity of sequence, our assembly achieves a higher BUSCO completion score (84%) than the existing goat reference assembly suggesting better quality annotation of gene models. Our results demonstrate that single-molecule sequencing can produce near-complete eukaryotic genomes at modest cost and minimal manual effort.

Transcript

1. From sequencing to chromosomes: new de
   novo assembly and scaffolding methods
   improve the goat reference genome
   Sergey Koren, @sergekoren
   Genome Informatics Section, NHGRI
   

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2. Hybrid error correction and de novo assembly
   of single-molecule sequencing reads
   Koren et al. (2012) Nature Biotechnology
   Reducing assembly complexity of microbial genomes
   with single-molecule sequencing
   Koren et al. (2013) Genome Biology
   Assembling Large Genomes with Single-Molecule Sequencing
   and Locality Sensitive Hashing
   Berlin et al. (2015) Nature Biotechnology
   With Canu:
   25x of PacBio P6C4 achieves:
   > 90% of bacteria assemble without gaps
   > QV40 (99.99%) consensus accuracy
   < 15 minutes of compute
   < $1,000 total cost
   Long read assembly
   

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3. 1 2 3 4 5 6 7 8 9 10 11 12
   13 14 15 16 17 18 19 20 21 22 X
   CHM1 Canu
   Human genome assembly solved?
   

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4. Contigs ≠ Genome
   ≠
   

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5. Goat PacBio vs RefV2
   PacBio RefV2
   Ctgs
   # bp 2.63Gbp 2.72Gbp
   # ctg 3,096 173,141
   Max 35,623,478 679,126
   N50 4,473,169 73,533
   Scfs
   # bp 2.63Gbp 2.69Gbp
   # scf 3,096 30
   Max 35,623,478 161,917,960
   N50 4,473,169 103,731,018
   

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6. ! CHIR 1.0 annotation LiftOver split mappings
   !  Example split gene
   ! Comparative mapping with sheep
   O. aries exons Exclusive PacBio Exclusive BGI Shared
   Total Mappings 9,534 564 225,365
   Unmapped 564 9,534 9,140
   Split Exons 592 1,930 1,528
   CHIR 1.0
   Sheep
   Antelope
   Cow
   Annotation comparison
   

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7. Algorithms Convert Images
   into Molecules
   Assembly Algorithms Align Molecules de novo for
   Constructing Consensus Genome Maps
   Cross-Mapping Across Multiple
   Samples or to a Reference
   High Throughput, High Resolution Imaging Gives Contiguous Molecules up to Mb Length
   •  Automated SV Detection
   •  Gap Sizing
   •  Genome Finishing
   Isolate
   High Molecular Weight DNA
   Label Specific
   Sequences Across
   the Entire Genome
   Transfer Labeled DNA into
   Cartridge for Scanning
   Load, Linearize & Image Labeled DNA in
   Repeated Cycling to Scan Whole Genome
   Insertion
   Customer Sample
   Irys Workflow for Genome Mapping
   Irys® Workflow- Overview
   © 2015 BioNano Genomics
   

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8. Goat PacBio+BioNano vs RefV2
   PacBio PacBio + BioNano RefV2
   Ctgs
   # bp 2.63Gbp 2.62Gbp 2.72Gbp
   # ctg 3,096 2,349 173,141
   Max 35,623,478 57,511,119 679,126
   N50 4,473,169 12,102,227 73,533
   Scfs
   # bp 2.63Gbp 2.62Gbp 2.69Gbp
   # scf 3,096 2,084 30
   Max 35,623,478 66,727,870 161,917,960
   N50 4,473,169 14,265,070 103,731,018
   

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9. chr1
   chr8 chrX
   chr28
   PacBio+BioNano to reference
   

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10. 3D model of
    genome
    Hi-C can be used for 3D modeling and scaffolding of
    genome assemblies
    Duan, et al. Nature, 2010
    Genome
    scaffolding
    Burton, et al. Nature Biotech, 2013
    1
    2
    3
    4
    5
    6
    7
    8
    9
    1 2 3 4 5 6 7 8 9
    chromosome
    chromosome
    From I. Liachko
    Haplotype
    phasing
    Selvaraj, et al. Nature Biotech, 2013
    Crosslink) Fragment) Proximity)
    Liga4on)
    Sequence)
    Junc4ons)
    

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11. Goat PacBio+BioNano vs RefV2
    PacBio PacBio + BioNano PacBio + BioNano + HiC RefV2
    Ctgs
    # bp 2.63Gbp 2.62Gbp 2.63Gb 2.72Gbp
    # ctg 3,096 2,349 1,522 173,141
    Max 35,623,478 57,511,119 66,489,255 679,126
    N50 4,473,169 12,102,227 23,340,314 73,533
    Scfs
    # bp 2.63Gbp 2.62Gbp 2.63Gbp 2.69Gbp
    # scf 3,096 2,084 525 30
    Max 35,623,478 66,727,870 157,517,791 161,917,960
    N50 4,473,169 14,265,070 91,787,174 103,731,018
    

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12. chr1
    chr8 chrX
    chr28
    PacBio+BioNano+HiC to reference
    

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13. chr1
    chr8 chrX
    chr28
    PacBio+BioNano+HiC+curation
    

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14. Acknowledgements
    !  Canu
    !  Adam Phillippy
    !  Brian Walenz
    !  Goat Project
    !  Derek M. Bickhart
    !  Adam M Phillippy
    !  Timothy P.L. Smith
    !  Shawn T. Sullivan
    !  Ivan Liachko
    !  Joshua N. Burton
    !  Maitreya J. Dunham
    !  Jay Shendure
    !  Alex R. Hastie
    !  Brian L. Sayre
    !  Heather J Huson
    !  George E. Liu
    !  Benjamin D. Rosen
    !  Steven G. Schroeder
    !  Curtis P. VanTassell
    !  Tad S. Sonstegard
    !  NHGRI
    ! Postdocs wanted!
    !  Genome Informatics Section
    !  Assembly
    !  Structural variation
    !  Infectious disease
    !  Undiagnosed disease
    !  http://www.genome.gov/27563366
    /MarBL
    

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15. PUBLIC DOMAIN NOTICE
    This presentation is "United States Government Work" under the
    terms of the United States Copyright Act. It was written as part of
    the authors' official duties for the United States Government and
    thus cannot be copyrighted. This presentation is freely available to
    the public for use without a copyright notice. Restrictions cannot
    be placed on its present or future use.
    Although all reasonable efforts have been taken to ensure the
    accuracy and reliability of the presentation and associated data,
    the National Human Genome Research Institute (NHGRI),
    National Institutes of Health (NIH) and the U.S. Government do
    not and cannot warrant the performance or results that may be
    obtained based on this presentation or data. NHGRI, NIH and the
    U.S. Government disclaim all warranties as to performance,
    merchantability or fitness for any particular purpose. Please cite
    the authors in any work or product based on this material.
    

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