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AGBT 2016 Workshop Magrini
This document summarizes sequencing and mapping plans and results for generating reference genomes. It discusses using PacBio to generate long reads, 10X Genomics for linked reads, and BioNano for physical mapping. Optimization of protocols was needed. Preliminary results showed the approaches provided complementary data to improve reference genomes, with each system having unique benefits and challenges. Further integration of the data sets could generate more robust reference genomes representing human genetic diversity.
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AGBT 2016 Workshop Magrini
- 1. MGI Reference Genomes Workshop VinceMagrini February 10th 2016
- 2. Sequencing Plan • PacBioLarge Insert Library Construction • Linked Reads with 10X Genomics • Physical Map contiguity using BioNano IRYS
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- 4. The NA19240 LargeInsert Library Experience 10,000 10,500 11,000 11,500 12,000 12,500 13,000 13,500 14,000 14,500 15,000 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 ROILength(bp) SMRT Cell ROI Length Lib4-ROI length Lib2-ROI length Lib3-ROI length Lib5-ROI length Lib6-ROI length Lib7-ROI length LibF-ROI length Lib8-ROI length
- 5. Considerations for PacBioWGS • High molecular weight genomic DNA • DNA must be of sufficient quality to allow for >30 kb shearing to produce PacBio Continuous Long Reads (CLR) • Consistent shearing >30 kb • Shearing genomic DNA >30 kb is challenging and requires a consistent technology • Preferred method: Diagenode Megaruptor • Alternate method: Covaris g-Tube • Sufficient DNA for PacBio sample prep • A single PacBio sample prep reaction requires 5 μg sheared DNA • One library is composed of 8-10 sample prep reactions • At least 2-4 libraries are required for 60x coverage
- 6. NA19240 Sheared DNAComparison Library Shear Type Shear Settings 2 g-Tube 5500 rpm 3 g-Tube 4800 rpm 4 g-Tube 4800 rpm 5 g-Tube 4500 rpm 6 MegaRuptor Menlo Park 30 kb 7 MegaRuptor Menlo Park 30 kb 8 MegaRuptor MGI 30 kb 30kb MGI 30kb MP G-Tube 4800 G-Tube 4500 ✜ ✪
- 7. PacBio Workflow DNA Shear DNARepair Ligation/Exonuclease BluePippin >18kb Sizing DNA Repair AMPure PB AMPure PB 3x AMPure PB Rinse wells AMPure PB AMPure PB Seq. Primer Anneal P6 Polymerase Bind MagBead Bind Sequencing 30 minutes or 4 hours 20 minutes to 2 hours Denature primer prior to use 4 to 6 hour collection time • Adding DNA Damage Repair after BluePippin sizing increased the average Reads of Insert length by ~1 kb. • Extending the P6 Polymerase Binding time from 30 minutes to 4 hours improved library complex loading per SMRT cell
- 8. Standard PacBio protocol(sample prep & complex) 0.0 200.0 400.0 600.0 800.0 1,000.0 1,200.0 1,400.0 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 ROIYield(Mbases) AverageROI(bp) SMRT Cell NA19240 Library 4 - Per SMRT Cell ROI length/yield ROI length (bp) ROI Yield (Mbases) Titration • No Post-BluePippin DNA Damage Repair • 30 min P6 polymerase bind 6 hour Movies 4 hour Movies 125 pM “on plate” loading concentration G-Tube 4800✜
- 9. DNA Damage Repair& extended P6 bind 0.0 200.0 400.0 600.0 800.0 1,000.0 1,200.0 1,400.0 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373839404142434445464748 ROIYield(Mbases) AverageROI(bp) SMRT Cell NA19240 Library 5 - Per SMRT Cell ROI length/yield ROI length (bp) ROI Yield (Mbases) • No Post-BluePippin DNA Damage Repair • 30 min P6 polymerase bind • Post-BluePippin DNA Damage Repair • 4 hour P6 polymerase bind G-Tube 4500✪
- 10. Menlo Park 30kb MegaRuptor 0.0 200.0 400.0 600.0 800.0 1,000.0 1,200.0 1,400.0 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 ROIYield(Mbases) AverageROI(bp) SMRT Cell NA19240 Library 7 - Per SMRT Cell ROI length/yield ROI length (bp) ROI Yield (Mbases) Titration 4hrP6bind 8Paclot# 231848 30minP6bind 8Paclot# 231848 4 hr P6 bind 8Pac lot # 231818 4 hr P6 bind 8Pac lot # 231848 4 hr P6 bind 8Pac lot # 231818 • Post-BluePippin DNA Damage Repair • 4 hour or 30 minute P6 polymerase bind 30kb MP
- 11. MGI 30 kbMegaRuptor 0.0 200.0 400.0 600.0 800.0 1,000.0 1,200.0 1,400.0 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 ROIYield(Mbases) AverageROI(bp) SMRT Cell NA19240 Library 8 - Per SMRT Cell ROI length/yield ROI length (bp) ROI Yield (Mbases) Titration 125 pM “on plate” loading concentration Clear cell-to-cell variability Failed cell 30kb MGI
- 12. PacBio NA19240 SequencingStatistics Sample 8 Packs Reads Mbp (Pol) RL Mbp(ROI) RL ROI Mbp/Cell NA19240 37 16,088,050 214,621 13,605 195,619 12,487 661 HG00733 30 15,858,313 209,619 13,193 190,430 11,958 793 HG00514 40 20,707,629 311,500 13,473 277,690 13,473 868 NA12878* 22 11,029,811 165,153 14,949 146,833 13,174 962 Assembly Stats will be highlighted in Tina’s presentation.
- 13. PacBio Sequencing Observations HG00514:4h v 6h movie lengths Instrument Movie Time Avg. ROI (bp) ROI Mb/Cell # Cells 00116 240 13,502 803 119 42274 240 13,036 881 95 00116 360 14,324 998 56 42274 360 13,282 1,063 24 • DNA Input and Sizing • The library DNA >18 kb is fractionated using the Sage Science BluePippin • DNA Damage Repair enzyme mix used post BluePippin (increased read length) • Chemistry • (+) DNA Damage Repair/4 hr bind: 970.2 Mbases/cell • Instruments • Longer average Reads • Increased Loading Efficiency • What about long term storage?
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- 15. Reconfigured Oligo - Usesinline index sequence - No P5 index – HiSeq X single index compatible 10X Genomics Overview
- 16. 10X Chromium Workflow WGXBeta Product Workflow gDNA Extraction GEM Formation Library Prep Long Ranger Pre-GEMs Post- GEMs NGS Loupe Qiagen MagAttract HMW Kit Qubit quantifications Dilute to 1 ng/ul gDNA egram Aliquot Master Mix NaOH template denature Load WGX Chip Run instrument Chip volume assessment Instrument log Isothermal incubation Emulsion breaking Bioanalyzer HiSeq X or HiSeq 4000 2x150bp sequencing 200pmol loading End Repair/A-tailing Adapter ligation SI PCR Bioanalyzer KAPA qPCR Visualization Demultiplexing Alignment De-duplication SNP and indel calling Large SV calling Phasing
- 17. • HiSeq 4000 •2x150, 200 pmol loading • 2 lanes Chromium NA19240 Library Sequencing Statistics Post Gem: Isothermal Amp size dist. Library Size Distribution The spike at 0 in that graph is due to the N's in the reference assembly.
- 18. NA19240 (MGI) NA12878(10X) Molecule Length (kb): 26,768 (±33,673) 94,923 (±64,103) DNA in Molecules > 10kb 50.85 % 95.0% DNA in Molecules > 100kb 1.38% 36.4% SNPs Phased: 99.1% 97.8% Longest Phase Block: 9.6 Mbp 34.7 Mbp N50 Phase Block: 1.9 Mbp 9.5 Mbp Chromium Molecule and Phasing Statistics
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- 20. Harvest Cells Dissociate Tissue Embed Cells inGel Plugs Lyse Cells, Digest Protein Melt and Digest Agarose Plugs Sample Cleanup Labeling Reaction BioNano Overview
- 21. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 10-500kb 100-500kb 150-500kb200-500kb 250-500kb >500kb 19240 19239 19238 19240 19239 19238 Mapped Molecule Quantity (Mb) 189,138.79 256,281.33 226,854.88 Mapped Avg Size (Kb) 232 280 289 Avg Label Density (per 100 Kb) 9.6 8.7 8.8 Number of Consensus Genome Maps 3051 2565 2798 Consensus Genome Maps Size (Mb) 2833.045 2965.972 2933.294 Consensus Genome Maps N50 (Mb) 1.276 1.685 1.477 Avg Depth of Mol Coverage 59.1 56.1 50.6 BioNano: Yoruban Trio Statistics Molecule Length Bin Molecules/Bin(%)
- 22. PacBio Assembly Contig BioNanoGenome Map Contigs Sequencing Plan Add 10X Linked Read information Add Dovetail Hi-C/chiCago Data
- 23. Summary • Goal: Generaterobust data sets for additional high-quality reference genome enhancing the full range of genetic diversity in humans • These long read (long range) sequencing/mapping applications vary in approach and will provide synergistic data sets to help accomplish our goal. • Each system possesses unique challenges and requires optimization of protocols and running conditions specific to our needs. • Experience and communication is key. • Increasing applications and utility • Polymerase read = read of insert • BAC Pooling • Low input SNV • Multicolor labeling
- 24. Acknowledgements The McDonnell GenomeInstitute at Washington University in St. Louis Rick Wilson Sean McGrath Amy Ly Ryan Demeter Dave Larson Karyn Meltz Steinberg Tina Graves Bob Fulton Derek Albracht Milinn Kremitzki Susan Rock Debbie Scheer Wes Warren Chad Tomlinson 10X Genomics Cassandra Jabara Michael Schnall-Levin Drew Kebbel Rob Tarbox Deanna Church BioNano Genomics Andrew Anfora Palak Sheth Alex Hastie Pacific Biosciences Paul Peluso Nick Sisneros
Editor's Notes
- #3 For this project, we are generating ~60X – 100X coverage of PacBio long read data based on Reads – of – insert data The plan: 1. de novo assembly of PacBio data. 2. scaffold the assembly with BioNano data as well as Dovetail chiCago and 10X genomics linked read data sets. With the combined data sets will begin to generate scaffolds and identify areas of f potential misassemblies. 3. We are also targeting difficult to assemble regions of the genome by sequencing BACs. Once the BACs are incorporated, we plan to align all of this data to the Reference very stringently to produce chromosomal AGPs. The end product will be a very high quality whole genome assembly. Our role in this project mostly focused on Larger Insert pacbio libraries, adding BioNano data sets, and early work with 10X genomics. Today, I will highlight our progress with these large molecule applications.
- #4 Highlight library – SMRT Bell Reads types Polymerase Reads Subreads Read on Insert Currently, we are enriching SMRT bell libraries > 18kb.
- #5 The sloppy slide For the NA19240 project, we generated a number of libraries (8). Please note, when I say library, I mean, for each library, a 10 reaction kit was used to Process 50µg of DNA in 8-10 independent reactions and then pooled. As this graph illustrates, the total number of SMRT cells for each of the libraries as well as the average ROI read length. Based on the these values, we Were able to consistently show inconsistent results; which required some tweaking of the process and many discussions with the Nick and Paul at PacBio. Based on these fruitful discussions, we were able to show a marked improvement which will be highlighted in this section.
- #7 Genomic DNA tape station shows and overlay of each of the different shearing conditions used to generated the multiple NA19240 SMRT Bell libraries The symbols represent the mode for each electropherograms. The table on the right highlights the shearing method for 7 of the 8 libraries, a
- #14 Data accumulation to date for each of the four genomes we’ve. The total number of cells is provide by the number of 8 packs, However, the table also illustrates the average number of ROI reads in Mbp per cell. Based on our modifications, we’ve transitioned the PacBio larger insert library protocol into production, And we are happy to report a positive trend with each new library – increased ROI data throughput per cell.
- #21 In addition to sequence based methods, we are also utilizing the the BioNano IRYS system to generate physical maps of the genome The advantage obtained with the physical allows us to maintain the order and orientation based on the nicking endonuclease recognition site. The slide illustrates the processing overview starting with a cell culture.
- #23 Another resource we have is a BioNano Genome Map of CHM1. BioNano is a nanopore mapping technology where the DNA in very long molecules is nicked and labeled and run through a nanochannel. Here is an example of the CHM1 Bionano map aligned to a 1.5 Mb Pb contig. On top in green is the PacBio contig. The lines indicate the in silico nick sites. The Blue bars indicate the Bionano contigs. You can see how well they align. The Bionano data can be used as an independent source to assess the CHM1.
- #25 I want to acknowledge all of the collaborators on this project and all of the work that has gone into it thus far.