Scaling up production of HMW DNA

Kelvin Liu, Circulomics

Transcript

1. Confidential Scaling Up Production of HMW DNA Dr. Kelvin Liu (kliu@circulomics.com) 1/16/2019
2. Confidential Long-Read Sequencing Requires Clean HMW DNA Precipitation and gel plug purification are slow and tedious. Poor DNA quality leads to short read lengths and low data yield.
3. Confidential Nanobind Magnetic Disks High surface area + low shear force + non-porous = immense binding capacity + high quality + high purity 100 μm 10 μm Magnetic Nanobind Disk Microscale Wrinkles Nanoscale Flakes Roll-to-Roll Manufacturing
4. Confidential Nanobind Protocol 1 hour bind, wash, and elute process parallels magnetic bead extraction. Step 1 Lyse Step 2 Bind Step 3 Wash Step 4 Elute Eluate with DNA Gentle Mixing Sample + lysis buffer Add Nanobind disk + isopropanol Discard supernatant + add wash buffer Discard supernatant + add water 2X Washes Magnetic Capture of Nanobind Magnetic Capture of Nanobind
5. Confidential Rapid HMW DNA Extraction (50 – 300+ kb) HMW protocols available for all sample types. Coast Redwood 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 582 kb L. monocytogenes S. enterica S. sonnei E. coli Whole Blood GM12878 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 582 kb 679 kb 776 kb 873 kb Nanobind CBB Big DNA Kit Available Now Nanobind Plant Nuclei Big DNA Kit Available Now 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 23 kb 9.4 kb 6.6 kb Human Muscle Nanobind Tissue Big DNA Kit Est. 1Q19
6. Confidential Rapid UHMW DNA Extraction (1+ Mb) Extract megabase-sized DNA in only 1 hr. CBB kit only. UHMW protocols for plants and tissues in development. Ladder 5.7 Mb 4.6 Mb 3.5 Mb Ladder UHMW 1 UHMW 2 UHMW 3 HMW 1 UHMW 1 UHMW 2 HMW 2 Ladder 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 582 kb 679 kb 776 kb 873 kb 970 kb
7. Confidential PacBio Sequel Nanobind HMW DNA generates exceptional data on PacBio Sequel. Sample Read Length N50 (bp) Max Read Length (bp) Total Data (Gb) GM12878 Cells 32,813 95,946 10.4 Escherichia coli 29,611 80,713 6.8 Shigella sonnei 35,251 83,705 9.0 Salmonella enterica 33,420 93,614 10.5 Listeria monocytogenes 32,091 95,382 10.2 Human Whole Blood 28,443 95,679 6.7 Coast Redwood 33,431 90,668 5.6 Rice 29,750 - 8.5 Brazilian Hyacinth 29,750 - 11.0 Data generated in collaboration with PacBio, Arizona Genomics Institute, and Timp Lab at JHU
8. Confidential Oxford Nanopore GridION Nanobind HMW DNA generates exceptional data on Oxford Nanopore MinION/GridION/PromethION. Sample Sequencing Read Length N50 (bp) Longest Read (bp) Yield (Gb) GM12878 Cells 5X NS, No SS, LSK109, 106C 34,417 253,002 5.8 Jurkat Cells No shear, No SS, LSK109, 106D 24,977 306,313 7.2 Escherichia coli 5X NS, 30 kb SS, LSK108, 106C 46,156 191,836 6.8 Listeria monocytogenes 5X NS, 30 kb SS, LSK108, 106C 48,391 254,985 8.0 Whole Blood No shear, No SS, LSK109, 106D 30,935 301,055 9.1 Coast Redwood 8 kb G-tube, No SS, LSK108, 106C 6,567 78,241 10.1 Coast Redwood 5X NS, No SS, LSK108, 106C 29,196 227,249 3.3 Data generated in collaboration with Timp Lab at JHU
9. Confidential ONT Ultra Long Reads Ultra long read sequencing using RAD004 kit is capable of reads up to 1.3 Mb. Longer reads possible with optimization of transposase DNA ratio. Nanobind Protocol Yield (Gb) Read Length N50 (bp) Reads >100 kb Longest Read (Mb) Reads >1 Mb Standard HMW 2.9 33,669 2809 .78 0 Modified UHMW 3.1 44,849 4698 1.1 2 Standard UHMW 1.9 60,734 3519 1.3 4 Std HMW 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 49 kb 97 kb 194 kb 291 kb 388 kb Mod UHMW 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb Std UHMW Data generated in collaboration with Timp Lab at JHU 0.1 1 10 100 1000 10000 0 500 1000 1500 Data (Mb) > Read Length Read Length (kb) Standard HMW Modified UHMW Standard UHMW
10. Confidential HMW + UHMW Tissue DNA Capable of obtaining large, high purity tissue from an array of both human and non-human tissues. 1 2 3 4 5 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 582 kb 679 kb 776 kb 873 kb 970 kb 6 7 8 Skate Testis Lynx Muscle 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 23 kb 9.4 kb 6.6 kb 1 2 3 4 5 Human Muscle 1 2 3 4 5 Human Kidney 6 23 kb 9.4 kb 6.6 kb 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 582 kb 679 kb 776 kb 1 2 3 4 Mouse Liver 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 582 kb Data generated in collaboration with VGL at Rockefeller University and DNA Sequencing Core at MPI
11. Confidential High Purity and Yield Differences in size, UV purity, yield, and ease of use can be seen across homogenization method used prior to Nanobind extraction. Sample 260/280 260/230 DNA (ng) / Tissue (mg) Human Muscle – TissueRuptor (L3) 1.86 2.08 246 Human Muscle – Dounce (L2) 1.88 1.95 363 Human Muscle – Pellet Pestle (L4) 1.90 1.68 321 Human Kidney – TissueRuptor (L3, L4) 1.81 1.97 294 Skate Testis – TissueRuptor (L3, L4) 1.85 2.29 1359 Lynx Muscle – TissueRuptor (L7, L8) 1.90 2.16 471 Mouse Liver – TissueRuptor (L3, L4) 1.86 2.09 925 Data generated in collaboration with VGL at Rockefeller University and DNA Sequencing Core at MPI
12. Confidential PacBio Sequel - Tissue Nanobind isolated HMW DNA from human skeletal muscle generates high N50 on PacBio Sequel. Nanobind Protocol Sequencing Subread N50 (bp) Max Subread (bp) Yield (Gb) Human Muscle – TissueRuptor 5X NS, 30 kb SS, 3.0 chemistry 36,369 218,345 14.8 Human Muscle – Dounce 5X NS, 30 kb SS, 3.0 chemistry 40,229 240,443 12.7 Human Muscle – Pellet Pestle 5X NS, 30 kb SS, 3.0 chemistry 32,132 204,903 12.4 0.1 1 10 100 1000 10000 100000 0 50 100 150 200 250 Subread Length (kb) Human Muscle - TissueRuptor Human Muscle- Dounce Human Muscle - Pellet Pestle Data (Mb) > Subread Length Data generated in collaboration with PacBio
13. Confidential Oxford Nanopore GridION - Tissue Nanobind isolated HMW DNA generates N50 approaching 50 kb and longest reads of almost 500 kb. Nanobind Protocol Sequencing Read Length N50 (bp) Longest Read (bp) Yield (Gb) Human Muscle – TissueRuptor 5X NS, No SS, LSK109, 106D 38,874 341,553 6.6 Human Muscle – Dounce 5X NS, No SS, LSK109, 106D 42,574 474,031 7.4 Mouse Liver – TissueRuptor 5X NS, No SS, LSK109, 106D 49,011 241,282 3.3 0.1 1 10 100 1000 10000 0 100 200 300 400 500 600 Read Length (kb) Human Muscle - TissueRuptor Human Muscle - Dounce Mouse Liver - TissueRuptor Data (Mb) > Read Length Data generated in collaboration with Timp Lab at JHU
14. Confidential PacBio Sequel – Colorado Potato Beetle Circulomics extracted Colorado Potato Beetle samples and sequenced on PacBio Sequel. 10-11 Gb per flow cell across 6 flow cells. Data generated in collaboration with Hawthorne Lab - University of Maryland PacBio Sequel - Colorado Potato Beetle Nanobind Protocol Polymerase N50 (bp) Subread N50 (bp) Max Subread Length (bp) Total Data (Gb) Beetle – Pellet Pestle 25,750 22,250 95,946 11.2 Nanobind Tissue Big DNA Kit Nanobind Protocol 260/280 260/230 DNA (ng) / Tissue (mg) Beetle – TissueRuptor 1.84 1.82 258.9 Beetle – Dounce 1.87 1.81 154.5 Beetle – Pellet Pestle 1.82 0.81 134.5 49 kb 97 kb 194 kb 291 kb 388 kb 485 kb 582 kb 679 kb 776 kb 873 kb 970 kb Pellet Pestle TissueRuptor Dounce
15. Confidential Automated HMW DNA Extraction Automated HMW Nanobind extractions can be run on existing automation instruments. Thermo Fisher KingFisher Scripts for cells, bacteria, and blood Liquid transfer instruments should be possible too High MW DNA 50 kb – 300+ kb UHMW megabase DNA may be possible. 200 µL Whole Blood Extraction DNA Yield (µg) 260/280 260/230 Thermo Fisher KingFisher (n=36) 11.1 ± 3.9 1.81 ± .02 1.50 ± .13 Manual (n=4) 9.7 ± 0.6 1.9 ± .01 1.9 ± .3 Comparable Manual and Automated Results Process samples in 30-40 minutes
16. Confidential Automated extractions sequence comparably to manual extractions on PacBio Sequel. Automated HMW Sequencing Validation Nanobind CBB Big DNA Kit – Automated Extractions – PacBio Sequel Sequencing Sample Subread N50 (bp) Max Subread Length (bp) Total Data (Gb) Manual GM12878 cells 32,813 95,946 10.4 Automated GM12878 cells 31,110 97,357 7.5 Manual E. coli 29,611 80,713 6.8 Automated E. coli 32,840 78,470 4.2 Manual Whole Blood 28,443 95,679 6.7 Automated Whole Blood 28,154 104,768 6.1 Data generated in collaboration with PacBio
17. Confidential Nanobind Extraction Throughput Rates of 125 samples per week can be achieved with current manual extractions. Even higher rates possible with automation. Method Cell/Bacteria/Blood Tissue Manual R&D Extractions 12 samples per hour 8 – 16 samples per 4 hours KingFisher DUO 12 samples per 40 min TBD KingFisher Flex 96 samples per 40 min TBD
18. Confidential Acknowledgements Questions? Jeffrey Burke Duncan Kilburn Renee Fedak Michelle Kim Kelvin Liu Rachael Workman Norah Hilger Winston Timp Heather Ferrao Brendan Galvin Keith Bjornson 1R43HG009859 2R44GM109618 2R44ES024180