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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

  1. 1. GIAB 2019 New Datasets
  2. 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. 3. ONT UL Reads
  4. 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. 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. 6. N50 and Throughput Over Time
  7. 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. 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. 9. Read Length and Coverage 15X Coverage by reads > 100Kb
  10. 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. 11. Acknowledgements ● Matt Loose Lab ● Nick Loman Lab ● Euan Ashley Lab ● Chunlin Xiao/NCBI ● JIMB ○ David Catoe ○ Noah Spies ○ Marc Salit
  12. 12. Nanopore Overview NIK SPENCER/NATURE
  13. 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. 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. 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

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