Telomere-to-telomere assembly of a complete human X chromosome
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Telomere-to-telomere assembly of a complete human X chromosome
- 1. Adam M. Phillippy Head, Genome Informatics Section Telomere-to-telomere assembly of a complete human X chromosome AGBT – March 2, 2019
- 2. • The human reference genome is incomplete • 368 unresolved issues, 102 gaps • Segmental duplications, rDNAs • Centromeres, telomeres, heterochromatin • These gaps contain important information • Missing reference sequence leads to analysis artifacts • Variation in these gaps is unexplored (e.g. rDNAs) • We don’t know what we don’t know… I have some troubling news…
- 3. @khmiga @aphillippy Karen Miga Adam Phillippy Let’s finish the human genome
- 4. • Repeats are long, reads are short • “If the overlap is of sufficient length to distinguish it from being a repeat in the sequence the two sequences must be contiguous.” • Rodger Staden, 1979, MRC Laboratory of Molecular Biology What’s the problem?
- 5. • The return of closed (bacterial) genomes • Bibersteinia trehalosi 192 Flashback to AGBT 2012
- 6. • How long are the repeats? • 7 kbp LINEs • 1 Mbp+ rDNA arrays • 1 Mbp+ centromere arrays • 10 Mbp+ heterochromatin blocks • Coverage and accuracy matter too • 1,000X of 100 bp reads at 100% accuracy? NO • 10X of 10,000,000 bp reads at 100% accuracy, YES • 100X of 100,000 bp reads at 90% accuracy, MAYBE? How long do reads need to be, for human?
- 7. • ONT R9 pore: E. coli CsgG membrane protein • Read lengths >1 Mbp possible Ultra-long nanopore sequencing *Assuming 3.4 Å per bp, 1 Mbp = 3,400,000 Å (0.34 mm) = 40,000x height of the pore 120 Å 85 Å 3.2 km in 37 m 8 cm
- 8. LONG READ CLUB Really very long reads indeed @pathogenomenick Nick Loman @mattloose Matt Loose
- 9. It’s time to finish the human genome CHM13 cell line from Urvashi Surti, Pitt; SKY karyotype from Jennifer Gerton, Stowers (N=46; XX) The Telomere-to-Telomere (T2T) consortium is an open, community-based effort to generate the first complete assembly of a human genome.
- 10. • 30x Nanopore ultra-long • Contig building • 60x PacBio • Polishing • 50x 10x Genomics • Polishing • BioNano • Structural validation We need long reads. Lots of long reads 100 kb
- 11. • Nanopore UL read length distribution is long tailed It pays to go deep repeat
- 12. • From May 1 – October 29, 2018 • 62 MinION/GridION flow cells • 8.9M reads, 98 Gb, 1.6 Gb / cell • N50 read length 76 kb • 44 Gb in reads >100 kb • Max read length 1.03 Mb • Assembled with Canu • 10x cov of 100 kb at 90% acc CHM13 sequencing Now upwards of 90 flow cells and counting…
- 13. The human genome, 2001 ref28 NG50 contig 0.5 Mbp
- 14. The human genome, 2019 CHM13 NG50 contig 75 Mbp (70x PacBio + 35x UL ONT) 13 14 15 16 17 18 19 20 21 22 X 1 2 3 4 5 6 7 8 9 10 11 12 Canu
- 15. The first complete assembly of a human chromosome
- 16. A complete X chromosome ddPCR
- 17. • Unique structural variants from PacBio • Unique k-mers confirmed by Duplex-Seq Stitching across the X centromere
- 18. An assembly is a hypothesis
- 19. Anchored 100 kb+ centromere reads Requires a careful measure of “mapping quality”
- 21. Centromere array validation 1.8 Mb 0.7 Mb 0.3 Mb
- 22. It’s time to finish the human genome
- 23. • Almost! • Have proven it’s possible for the X chromosome • T2T assembly of all chrs within the next 2 years • Remaining challenges • Satellite arrays, rDNA arrays, segmental duplications • Nanopore consensus quality • Targeted long-read sequencing • Better methods for phasing repeats and haplotypes Are we there yet?
- 24. • github.com/nanopore-wgs-consortium/chm13 • Draft whole-genome assemblies • Nanopore ultra-long reads • 10x Genomics reads • BioNano DLS (WashU) • PacBio (SRA) • Coming soon: • Hi-C (Arima Genomics) All our CHM13 data is openly released
- 25. NHGRI • Sergey Koren • Arang Rhie • Jim Mullikin • Alice Young • Shelise Brooks • Valerie Maduro • Gerard Bouffard • Sofia Barreira • Andy Baxevanis • Nancy Hansen • Karen Miga, UCSC • Jennifer Gerton, Stowers • Tamara Potapova, Stowers • Tina Graves Lindsay, WashU • Ira Hall, WashU • Valerie Schneider, NCBI • Kerstin Howe, Sanger • Jo Wood, Sanger • Matt Loose, Nottingham • Nick Loman, Birmingham • Urvashi Surti, Pitt (ret.) Acknowledgements