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Generating high-quality human reference genomes using PromethION nanopore sequencing
- 1. Generating high-quality human referencegenomes using PromethION nanopore sequencing Karen Miga khmiga
- 2. One (haploid) genomereference assembly for all of humanity
- 4.
- 5. Deletion Large Structural Variation Insertion SNVComputational Pan-Genomics TEAM
- 6. Deletion Insertion SNV Large Structural Variation Computational Pan-Genomics TEAM Samplingto maximize allelic diversity
- 7. Analysis: Adam Phillippy(NIH/NHGRI), Fritz Sedlazeck (Baylor), Benedict Paten (UCSC) Sampling to maximize allelic diversityTop 10 Individuals Selected to Maximize Diversity
- 8. Sampling to maximize allelicdiversity Emphasis on Phasing: Selection of Trios Top 10 Individuals Selected to Maximize Diversity Emphasis on Quality: Low Passage Cells Analysis: Adam Phillippy (NIH/NHGRI), Fritz Sedlazeck (Baylor), Benedict Paten (UCSC)
- 9. High-quality, phased reference genomesTop10 Individuals Selected to Maximize Diversity Analysis: Adam Phillippy (NIH/NHGRI), Fritz Sedlazeck (Baylor), Benedict Paten (UCSC) Adam Phillippy (NIH/NHGRI) High Coverage PacBio Genome Assemblies From the Same Individuals Collaboration
- 10. Dramatic increase inthe production of high quality reference assemblies Long read Sequencing Compute: Assembly + Technology Bottleneck Computational Pan-Genomics TEAM Sampling to maximize allelic diversity
- 11. Long Read Sequencing Consistencyin Assembly Quality Capacity to Scale: Parallelized Long-Read Sequencing Comprehensive Genome Representation 1 2 3
- 12. Long Read Sequencing Consistencyin Assembly Quality Capacity to Scale: Parallelized Long-Read Sequencing PromethION Comprehensive Genome Representation 1 2 3 100 kb+ Reads Scalable Assembly Tools Multi-flow Cells
- 13. Sequencing 11 ReferenceGenomes in 9 Days
- 14. Flip Flop Racon Medakawtdbg2 HiRise Sequencing/ BasecallingAssembly Polishing Scaffolding 4x FINISHED ASSEMBLY HiC Data Phasing
- 15. https://www.circulomics.com/ Centrifuge Wash Step Re-suspend Size-selected HMWDNA gDNA + buffer x2 Short Read Eliminator Kit Sequencing strategy for enrichment of long reads Decrease Standard HMW DNA Prep Circulomics Short Read Eliminator Kit Increase Read Lengths (kb) NumberofBases(Mb)
- 16. https://www.circulomics.com/ Centrifuge Wash Step Re-suspend Size-selected HMWDNA gDNA + buffer x2 Short Read Eliminator Kit FoldEnrichment Read Lengths (kb) Sequencing strategy for enrichment of long reads Enrichment of 100kb+ reads 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Read Lengths (kb) NumberofBases(Mb)
- 17. https://www.circulomics.com/ Centrifuge Wash Step Re-suspend Size-selected HMWDNA gDNA + buffer x2 Short Read Eliminator Kit FoldEnrichment Read Lengths (kb) Sequencing strategy for enrichment of long reads Enrichment of 100kb+ reads 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Read Lengths (kb) 0 5 10 15 20 HG020HG02055HG01243HG01109HG00733GM24385GM24149GM24143 Coverage 7x 5x >10 kb 100 kb+ Boost in Overall Coverage of 100kb+ NumberofBases(Mb)
- 18. 30 35 40 45 50 24143 24149 2438500733 01109 01243 02055 02080 02723 03098 03492 0 1000 2000 3000 4000 5000 6000 7000 8000 100kb+ 10-100kb <10kbMb Read Len N50s: 44kb Sequencing strategy for enrichment of long reads 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 20 40 60 80 220200180160140120100 Read Length (kb) NumberofBases(Gb)
- 19. GM24143 GM24149 GM24385 HG00733 HG01109 HG01243 HG02055 HG02080 HG02723 HG03098 HG03492 0 30 6090 Diploid Genomes min max 62 79 80 71 68 74 79 81 71 107 98 45 40 68 41 64 43 52 62 27 82 88 Flow Cell Throughput (Gb) ave 69 Gb Per Flow Cell Sequencing Throughput
- 20. GM24143 GM24149 GM24385 HG00733 HG01109 HG01243 HG02055 HG02080 HG02723 HG03098 HG03492 0 30 6090 Diploid Genomes min max 62 79 80 71 68 74 79 81 71 107 98 45 40 68 41 64 43 52 62 27 82 88 Flow Cell Throughput (Gb) ave 69 Gb Per Flow Cell 48x 54x 69x 52x 61x 57x 61x 74x 47x 83x 85x cov 159 (Gb) 177 227 173 201 188 201 243 156 274 280 Total throughput 100 kb+ Reads (ave 22Gb, 7.3x)
- 21. 02055 01109 2438503492 24143 02723 01243 24149 02080 00733 03098 Individuals SequenceIdentity(GRCh38) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 Flip-flop Non-flip flop HG00733 Flow Cell Replicates Evaluation of Read Accuracy 0.5 0.6 0.7 0.8 0.9 1.0 0.5 0.6 0.7 0.8 0.9 1.0 Sequence Identity Sequence Identity mode: 0.915 median: 0.88203
- 22. Flip Flop Racon Medakawtdbg2 HiRise Sequencing/ BasecallingAssembly Polishing Scaffolding 4x FINISHED ASSEMBLY HiC Data Phasing Assembly Performance: Base Accuracy
- 23. Flip Flop Racon Medakawtdbg2 HiRise Sequencing/ BasecallingAssembly Polishing Scaffolding 4x FINISHED ASSEMBLY HiC Data Phasing Assembly Performance: Base Accuracy HG00733 99.18% 2.76 GB aligned Consensus Base Accuracy (GRCh38)
- 24. Flip Flop Racon Medakawtdbg2 HiRise Sequencing/ BasecallingAssembly Polishing Scaffolding 4x FINISHED ASSEMBLY HiC Data Phasing Assembly Performance: Base Accuracy HG00733 99.18% 2.76 GB aligned Consensus Base Accuracy (GRCh38) • Not phased alignments • Additional polishing steps (pilon/ methylation aware polishing) • Alignments are not to the individuals genome
- 25. Flip Flop Racon Medakawtdbg2 HiRise Sequencing/ BasecallingAssembly Polishing Scaffolding 4x FINISHED ASSEMBLY HiC Data Phasing Assembly Performance: Base Accuracy HG00733 99.18% 2.76 GB aligned Consensus Base Accuracy (GRCh38) • Alignments are not to the individuals genome 0.9976 Complete BAC alignments 21 BACs: 3.1Mb
- 26. Flip Flop Racon Medakawtdbg2 HiRise Sequencing/ BasecallingAssembly Polishing Scaffolding 4x FINISHED ASSEMBLY HiC Data Phasing Assembly Performance: Base Accuracy HG00733 99.18% 2.76 GB aligned Consensus Base Accuracy (GRCh38) • Alignments are not to the individuals genome Complete BAC alignments 21 BACs: 3.1Mb 0.9976 NA12878 ONT (NBT 2018, update): Nanopolish (x2), CpG methylation-mode (Sergey Koren and Adam Phillippy) *
- 27. Assembly Performance: Contiguity Workflowconsistency Contigsize(Mb)
- 28. Assembly Performance: Contiguity+ HiC Scaffoldsize(Mb)
- 29. Assembly Performance: SpanningRepeats Test against a benchmark set of GRCh38 segmental duplications SD Mid-Range (25kb-100kb): Large (100kb+): 1. No assembly gaps (50kb+/-) 2. Flanking unique sequences (10 kb +/-) 146 Sites 121/122 Regions 23/24 Regions Assembly spans: 98.6% 5’ 3’
- 30. 10 Reference GenomeAssemblies in 10 Days
- 31. Production Cost Estimate: $10,000/Genome Sequencing 10Reference Genomes in 10 Days Long read Sequencing Compute: Assembly +
- 32. Flip Flop Racon Medakawtdbg2 HiRise Sequencing/ BasecallingAssembly Polishing Scaffolding 4x FINISHED ASSEMBLY HiC Data Phasing Not yet running at full capacity Improvement Assembly and Polishing: Reduce cost — Improve quality Haplotype Phasing
- 33. Deletion Insertion SNV Large Structural Variation Computational Pan-Genomics TEAM Samplingto maximize allelic diversity Production of high quality reference assemblies
- 34. Benedict Paten David Haussler EdGreen Mark Akeson Sofie Salama TEAM Miten Jain Hugh Olsen Kristof Tigyi Marina Haukness Ryan Lorig-Roach Trevor Pesout Joel Armstrong Nicholas Maurer UCSC Adam Novak Glenn Hickey Jordan Eizenga Erik Garrison Jean Monlong Xian Chang Adam Phillippy (NIH/NHGRI) Fritz Sedlazeck (Baylor) Simon Mayes Vania Costa Daniel Garalde David Stoddart Rosemary Dokos Jon Pugh Chris Seymour Chris Wright ONT Kelvin Liu Duncan Kilburn Paolo Carnevali