New data from giab genomes intro and ultralong nanopore
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New data from giab genomes intro and ultralong nanopore
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New data from giab genomes intro and ultralong nanopore
- 2. 2019 Datasets ● Oxford Nanopore ○ Ultra-Long Read (HG002) - Nate Olson ○ Promethion (AJ and HC Trio) - Karen Miga ● Pacific Biosciences ○ Sequel (HC Trio) https://doi.org/10.1101/562611 ○ Sequel II 10 and 15kb CCS (HG002) - Aaron Wenger (https://doi.org/10.1101/519025) ● Strand-seq - Peter Lansdorp
- 3. ONT UL Reads
- 4. UL Read Sample Prep Challenges Concentrated clean high MW DNA is hard to get! ● High MW DNA is very susceptible to shearing ● DNA nicking results in shorter reads ● Highly concentrated DNA is very viscous
- 5. Grow and Pellet Human Cells Use FreshFreeze Embed in Agarose Lyse in Liquid Lysis Buffer Embed in Agarose Lyse in Liquid Lysis Buffer Dialysis PCIA/ EtOH PCIA/ EtOH Dialysis Dialysis Pellet and Resuspend PCIA/ EtOH Dialysis Dialysis Pellet and Resuspend Dialysis PCIA/ EtOH Dialysis Pellet and Resuspend Dialysis Pellet and Resuspend Filtration Filtration JIMB Sample Prep Method Optimization
- 6. N50 and Throughput Over Time
- 7. Grow and Pellet Human Cells Use FreshFreeze Embed in Agarose Lyse in Liquid Lysis Buffer Embed in Agarose Lyse in Liquid Lysis Buffer Dialysi s PCIA/ EtOH PCIA/ EtOH Dialysis Dialysis Pellet and Resuspend PCIA/ EtOH Dialysi s Dialysis Pellet and Resuspend Dialysi s PCIA/ EtOH Dialysis Pellet and Resuspend Dialysis Pellet and Resuspend Filtration Filtration JIMB Optimized Protocol
- 8. Dataset Characteristics ● 111 Total Flowcells ○ 84 MinION ○ 25 GridION ○ 2 PromethION ● > 11 M Reads ● 47 Longer than 1 Mb ● Longest read > 1.5 Mb
- 9. Read Length and Coverage 15X Coverage by reads > 100Kb
- 10. Conclusions ● Data availability ● What we plan to do with the data - utility in benchmark set development ○ Call and Confirm SVs ○ Call small variants in difficult to map regions ● Manuscript Plan ○ SVs, small variants, assembly, error correction
- 11. Acknowledgements ● Matt Loose Lab ● Nick Loman Lab ● Euan Ashley Lab ● Chunlin Xiao/NCBI ● JIMB ○ David Catoe ○ Noah Spies ○ Marc Salit
- 13. MinION • One flowcell • Up to 512 nanopores at once • One sample port per flowcell • 10-20 GB of data PromethION • Up to 48 flow cells • Up to 3000 nanopores at once • 4 sample ports per flowcell • up to 12 TB in 48 hours nanoporetech.com GridION • 5 X MinION Flowcells
- 14. Protocol that has consistently yielded highest N50 and greatest throughput: • Lyse cells in Liquid buffer with RNase A • Digest Proteins with Proteinase K • Purify DNA using PCIA • Concentrate and further purify by EtOH precipitation/hooking • pellet DNA on Benchtop MiniFuge (~2500 g) for 5 seconds • load ~ 15 ug with 1.5 uL Transposase • Mix painfully slowly at each step of library prep by SLOWLY dialing the pipette, not pushing the plunger, in order to pipette sample up and down 4 times • Restart runs when pore number decreases by roughly half
- 15. Overview of GIAB Data ● GIAB samples have been characterized using ○ ## Sequencing Platforms ○ ## Mapping Technologies ○ For complete list - https://github.com/genome-in-a-bottle/giab_data_indexes ● GIAB Data Release Policy