Theorem: Long reads solve everything Proof Everything is a metagenome Assembly solves metagenomics Long reads solve assembly Long reads solve everything * *This is hyperbole
! Want to find an optimal tour ! Consistent with the original fragments ! Fragment arrival rate is consistent with a Poisson process ! Read-pair distance and orientation is preserved, etc… ! Many possible paths ! Use a variety of heuristics to find good path ! Output only the high-confidence paths through the graph (contigs) ! Hard to assemble, easier to validate ! Run a bunch and pick the best! Mark Chaisson Short read assembly is hard Automated ensemble assembly and validation of microbial genomes. Koren et al. (2014) BMC Bioinformatics
! Repeats cause tangles and cycles in the graph ! A randomly generated sequence is trivial to assemble ! Repeats shorter than the read length don’t matter ! “It” ! “It was” ! “It was the best” ! “It was the best of times” ! “With his hands in his pockets” Why is assembly hard? >1,000 SSR 320 TE 2 SegDup 1 Unique 3 Meta
How long are microbial repeats? Golden Threshold Reducing assembly complexity of microbial genomes with single-molecule sequencing. Koren et al. (2013) Genome Research
k = 1,000 k = 7,000 Golden Threshold P5C3 (84% > 7Kbp) How long do reads need to be? k = 50 One chromosome, one contig. Koren and Phillippy (2015) Curr Opin Microbiol
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
Improvements in: Y heterochromatin Telomeres, centromeres TE and repeat resolution New biology discovered! Complete assembly of Chromosome 3L D. melanogaster Assembly
Birth of a new gene on the Y chromosome of Drosophila melanogaster Carvalho et al. (2015) Finally, we emphasize the utility of PacBio technology in dealing with difficult genomic regions: as was the case with the Mst77Y region, [MHAP] produced a seemingly error-free assembly of the FDY region, something that has eluded us for years of hard work. Here we describe flagrante delicto Y (FDY), a very young gene that shows how Y-linked genes were acquired. FDY originated 2 million years ago from a duplication of a contiguous autosomal segment of 11 kb containing five genes that inserted into the Y chromosome. Four of these autosome-to-Y gene copies became inactivated (“pseudogenes”), lost part of their sequences, and most likely will disappear in the next few million years. FDY, originally a female- biased gene, acquired testis expression and remained functional.
Genetic variation and the de novo assembly of human genomes Chaisson, Wilson, Eichler (2015) emphasize the importance of complete de novo assembly as opposed to read mapping as the primary means to understanding the full range of human genetic variation.
Bridging the gap From http://www.bionanogenomics.com/technology/irys-technology/ Bionano Genomics HiC Crosslink) Fragment) Proximity) Liga4on) Sequence) Junc4ons) adapted from Lieberman-Aiden, et. al. Science, 2009
3D model of genome Hi-C can be used for 3D modeling and scaffolding of genome assemblies Duan, et al. Nature, 2010 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) Genome scaffolding
Conclusions & Future Work ! Centromeres/telomeres remain a challenge ! HiC fails due to low interactions ! Bionano fails due to missing restriction sites ! Improved algorithms to combine technologies ! Assembler output ! Assembling populations ! Graph based assembly and formats ! Long read polishing and phasing
Acknowledgements ! Canu ! Adam Phillippy ! Brian Walenz ! MHAP ! Konstatin Berlin ! Goat ! 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 ! Dermatofibrosarcoma ! Alejandro Gutierrez ! Sarah Morton ! Mike Schatz ! Maria Nattestad ! Fritz Sedlazeck ! A. gambiae ! Andrew Hall ! Philippos-Aris Papathanos ! Atashi Sharma ! Changde Cheng ! Omar Akbari ! Lauren Assour ! Nicholas Bergman ! Alessia Cagnetti ! Andrea Crisanti ! Tania Dottorini ! Elisa Fiorentini ! Roberto Galizi ! Jonathan Hnath ! Xiaofang Jiang ! Tony Nolan ! Diana Radune ! Maria Sharakhova ! Aaron Steele ! Vladimir A. Timoshevskiy ! Nikolai Windbichler ! Simo Zhangl ! Matthew W. Hahn ! Scott J. Emrich ! Igor V. Sharakhov ! Zhijian Tu ! Nora J. Besansky ! D. melanogaster ! Casey Bergman ! Sue Celniker ! Jason Chin ! Jane Landolin ! NHGRI ! Postdocs wanted! ! Genome Informatics Section ! Assembly ! Structural variation ! Infectious disease ! Undiagnosed disease ! http://www.genome.gov/27563366 /MarBL
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