Telomere-to-telomere assembly of a complete human chromosomes
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1. Karen Miga presented on reaching complete telomere-to-telomere chromosome assemblies using long-read sequencing. 2. The current human reference genome is incomplete, with gaps and unresolved issues remaining. 3. Miga's lab generated a high-quality assembly of chromosome 21 for the CHM13 genome using Oxford Nanopore long reads totaling 155 GB of sequence data.
Telomere-to-telomere assembly of a complete human chromosomes
- 1. Telomere-to-telomere assembly of a complete human chromosomes Karen Miga UC Davis Genetics Seminar Sept 30, 2019 @khmiga
- 2. New Era in Genetics and Genomics We are finally reaching complete, high-quality telomere-to-telomere chromosome assemblies
- 3. New Era in Genetics and Genomics We are finally reaching complete, high-quality telomere-to-telomere chromosome assemblies Human reference genome is incomplete. • 368 unresolved issues, 102 gaps • Segmental duplications, gene families, satellite arrays, centromeres, rDNAs • Uncharacterized sequence variation in the human population
- 4. New Era in Genetics and Genomics We are finally reaching complete, high-quality telomere-to-telomere chromosome assemblies Human reference genome is incomplete. • 368 unresolved issues, 102 gaps • Segmental duplications, gene families, satellite arrays, centromeres, rDNAs • Uncharacterized sequence variation in the human population chr21
- 5. New Era in Genetics and Genomics We are finally reaching complete, high-quality telomere-to-telomere chromosome assemblies Human reference genome is incomplete. • 368 unresolved issues, 102 gaps • Segmental duplications, gene families, satellite arrays, centromeres, rDNAs • Uncharacterized sequence variation in the human population Our current understanding of genome biology and function30 Mb chr21
- 6. New Era in Genetics and Genomics We are finally reaching complete, high-quality telomere-to-telomere chromosome assemblies Human reference genome is incomplete. • 368 unresolved issues, 102 gaps • Segmental duplications, gene families, satellite arrays, centromeres, rDNAs • Uncharacterized sequence variation in the human population Our current understanding of genome biology and function30 Mb chr21 ~20 Mb ?
- 7. Challenge: Generating assemblies across repetitive regions that span hundreds of kilobases. Repeats (100 kb+) Unique variant Unique variant Can high-coverage ultra-long sequencing resolve complete assemblies of the human genome?
- 9. It’s time to finish the human genome The Telomere-to-Telomere (T2T) consortium is an open, community-based effort to generate the first complete assembly of a human genome.
- 10. Our target: CHM13hTERT Cell line from Urvashi Surti, Pitt; SKY karyotype from Jennifer Gerton and Tamara Potapova, Stowers N=46; XX
- 11. Our target: CHM13hTERT Cell line from Urvashi Surti, Pitt; SKY karyotype from Jennifer Gerton and Tamara Potapova, Stowers N=46; XX
- 12. Intramural Sequencing Center CHM13 Sequencing 94 MinION/GridION flow cells 11.1M reads 155 Gb (1.6 Gb / flow cell) (50x) 99 Gb in reads >50 kb (32x) 78 Gb in reads >70 kb (25x) Max mapped read length 1.04 Mb From May 1/18 – Jan 8/19
- 13. Intramural Sequencing Center CHM13 Sequencing 94 MinION/GridION flow cells 11.1M reads 155 Gb (1.6 Gb / flow cell) (50x) 99 Gb in reads >50 kb (32x) 78 Gb in reads >70 kb (25x) Max mapped read length 1.04 Mb From May 1/18 – Jan 8/19 50x Nanopore ultra-long Contig building 60x PacBio Polishing 50x 10x Genomics Polishing BioNano Structural validation
- 14. • 2.94 Gbp assembly NG50: 75 Mbp • Exceeds the continuity of the reference genome GRCh38 (56 Mbp NG50 contig size). • Subset of chromosome assemblies break only at centromere. Roadmap for completing the genome Canu
- 15. Canu
- 16. Canu
- 17. Orthogonal Validation Jo and Valerie
- 19. 2.2 - 3.7 Mb mean of 3010 kb (S.D. = 429; n = 49)
- 21. STRUCTURAL VARIANT 151516 15 3 8 2 8 4 Assemble contigs Using overlapping SV patterns
- 22. XqXp Scaffold Assembly of XCEN
- 23. XqXp Rel3 Assembly: ~3.1 Mb The assembly is a hypothesis(!)
- 24. 2107 294659 Beth SullivanJennifer Gerton Edmund Howe Rel3 Assembly: ~3.1 Mb
- 25. @NanoporeConf | #NanoporeConf Marker-assisted mapping Adam Phillippy Arang Rhie Sergey Koren
- 26. @NanoporeConf | #NanoporeConf Create a scaffold of unique, or single copy k-mers genome-wide Marker-assisted mapping Adam Phillippy Arang Rhie Sergey Koren Marker-assisted mapping
- 27. @NanoporeConf | #NanoporeConf Anchor high-confident long-read alignments to repeat assemblies Marker-assisted mapping Adam Phillippy Arang Rhie Sergey Koren Marker-assisted mapping
- 28. 28 Confident mapping of long reads using a single-copy k-mer strategy Identify and mark all sites of unique anchors across the chromosome chrX • 21-mers that appear ~c times in Illumina data • Also found in PacBio/Nanopore reads • Less frequent in the centromere, but still there • (Validated with Duplex-Seq)
- 29. 29 Confident mapping of long reads using a single-copy k-mer strategy Filter long read alignments: retaining those with unique k-mer anchoring chrX chrX
- 30. 30 Spacing of single-copy k-mers can be irregular in repeat-dense regions chrX chrX X CENTROMERE ARRAY CENTROMERE CENX: 3.1 Mbps Number of k-mers: 2,034 Spacing N50: 6,879 Longest distance between k-mers : 53,798 bp
- 31. 31 10XG Polishing Unique K-mer-based filtering: Nanopore Reads longranger + freebayes (two rounds) nanopolish (two rounds) arrow (two rounds) Unique K-mer-based filtering: PacBio (CLR) Reads chrX chrX chrX
- 32. GAGE pre-polishing ChrX GAGE array: 19 tandemly arrayed ~9.4 kb repeats Coverage 250 200 150 100 50 0 Base position Most frequent base Second most frequent base (error) 19 tandemly arrayed ~9.4 kb repeats
- 33. GAGE with marker-assisted polishing Most frequent base Second most frequent base (error) ChrX GAGE array: 19 tandemly arrayed ~9.4 kb repeats Coverage 250 200 150 100 50 0 Base position 19 tandemly arrayed ~9.4 kb repeats
- 34. 34 CSS/HiFi Evaluation chrX HiFi Alignments to Evaluate Polishing CENTROMERE X: BEFORE POLISHING DXZ1: 3.1 Mb
- 35. 35 CSS/HiFi Evaluation chrX HiFi Alignments to Evaluate Polishing CENTROMERE X: AFTER POLISHING NOTE: Underlying satellite array structure remains the same. DXZ1: 3.1 Mb
- 36. Opens the whole genome to analysis Ariel Gershman Winston Timp’s Laboratory
- 40. 1. Structurally validated assembly from telomere-to-telomere. Including 3.1 Mb tandem repeat at the X centromere and providing a complete assessment across tandemly repeated gene families. Finished T2T X Chromosome: High Accuracy and High Continuity
- 41. 1. Structurally validated assembly from telomere-to-telomere. Including 3.1 Mb tandem repeat at the X centromere and providing a complete assessment across tandemly repeated gene families. 2. Novel polishing strategy capable of improving the quality of large repeat- rich regions. Demonstrating dramatic improvements in quality over the entirety of the X chromosome. Finished T2T X Chromosome: High Accuracy and High Continuity
- 42. 1. Structurally validated assembly from telomere-to-telomere. Including 3.1 Mb tandem repeat at the X centromere and providing a complete assessment across tandemly repeated gene families. 2. Novel polishing strategy capable of improving the quality of large repeat- rich regions. Demonstrating dramatic improvements in quality over the entirety of the X chromosome. 3. Statistics of CHM13 full length BAC alignments to polished assembly: 275/341 (81%) QV 37.4 QV 27.9 153/341 (45%) QV 37.7 QV 27.4 Vollger M, Logsdon, G et al. bioRxiv doi.org/10.1101/635037 MeanMedianBACs Aligned HiFi UL-asm Finished T2T X Chromosome: High Accuracy and High Continuity
- 43. @NanoporeConf | #NanoporeConf It is time to finish the human genome
- 44. • github.com/nanopore-wgs-consortium/chm13 • 120x Nanopore reads • NHGRI, UW, Nottingham, • UC Davis (PromethION, Megan Dennis) • 50x 10x Genomics linked reads (NHGRI) • 70x PacBio CLR reads (WashU) • 24x PacBio HiFi reads (UW) • 40x Hi-C (Arima Genomics) • BioNano optical map (WashU) • Unpolished Canu assemblies NEW! Rel3 open data release
- 45. Additional ultra-long ONT data from Glennis Logsdon (UW) Read length Coverage Percent of data >50 kbp 12X 86% >100 kbp 9.1X 66% >150 kbp 6.8X 49% >200 kbp 4.9X 35% >250 kbp 3.4X 24% N50 = 147.1 N1 = 649.6 Max = 1538.3 0.1 1 10 100 1000 10,000 Read length (kbp) 20,000 17,500 15,000 12,500 10,000 7,500 5,000 2,500 0 Numberofreads 13.9X coverage • github.com/nanopore-wgs-consortium/chm13
- 46. • Minimal change in continuity • 79.5 Mbp (rel2) vs. 71.8 Mbp (rel3) NG50 • Don’t judge assemblies based on continuity • Tricky regions are fixed • GAGE and more SegDups automatically resolved • Improved BAC validation • 288 (rel2) vs. 310 (rel3) of 341 BACs resolved • 1 chromosome down, 23 to go… Triple the coverage, what changed?
- 47. Goal of a complete human genome in the next two years. Challenges in front of us: • Acrocentric p-arms • Large segmental duplications • Classical Human satellites 2,3 Establishing new benchmarking standards (XChr) Pioneering new pipelines: Polishing, repeat assembly, and array structural validation. Setting the bar higher for quality and completeness.
Editor's Notes
- #8: KEY POINT HERE: spacing of unique variants… Some regions are easier than others….
- #31: Number of k-mers: 2,034 Spacing N50: 6,879 Longest distance: 53,798 bp
- #41: Median BAC QV 37.4 (mean QV 28.0) vs median QV 37.6 (mean WV 27.4 ) for the best CHM13 HiFi asm. And resolve 85% of BACs at >99.8% idy v.s. 54% for prior PacBio asm. T otal BACs: 341 Compressed: 166 1 Median: 99.9895 QV: 39.78811 Mean: 99.8706 QV: 28.88052 Mitchell HiFi: 153 1 Median: 99.9827 QV: 37.61954 Mean: 99.81871 QV: 27.41627 UL + 10x: 275 1 Median: 99.982 QV: 37.44727 Mean: 99.84145 QV: 27.99832
- #42: Median BAC QV 37.4 (mean QV 28.0) vs median QV 37.6 (mean WV 27.4 ) for the best CHM13 HiFi asm. And resolve 85% of BACs at >99.8% idy v.s. 54% for prior PacBio asm. T otal BACs: 341 Compressed: 166 1 Median: 99.9895 QV: 39.78811 Mean: 99.8706 QV: 28.88052 Mitchell HiFi: 153 1 Median: 99.9827 QV: 37.61954 Mean: 99.81871 QV: 27.41627 UL + 10x: 275 1 Median: 99.982 QV: 37.44727 Mean: 99.84145 QV: 27.99832
- #43: Median BAC QV 37.4 (mean QV 28.0) vs median QV 37.6 (mean WV 27.4 ) for the best CHM13 HiFi asm. And resolve 85% of BACs at >99.8% idy v.s. 54% for prior PacBio asm. T otal BACs: 341 Compressed: 166 1 Median: 99.9895 QV: 39.78811 Mean: 99.8706 QV: 28.88052 Mitchell HiFi: 153 1 Median: 99.9827 QV: 37.61954 Mean: 99.81871 QV: 27.41627 UL + 10x: 275 1 Median: 99.982 QV: 37.44727 Mean: 99.84145 QV: 27.99832