New data from giab genomes intro and ultralong nanopore

1. GIAB 2019 New
Datasets

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

12. Nanopore Overview
NIK SPENCER/NATURE

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