Meeting with Andrea Quattrini, advisor for SIFP, and Dan MacGuigan, collaborator who has worked a lot with Nanopore sequencing (MinION and PromethION)
Major Questions:
Dealing w fungal contamination?
Adaptive sampling?
How many samples can we multiplex?
Ligation vs rapid sequencing?
Notes:
Smithsonian has a MinION!
flow cells have pretty short shelf life, so even if other Smithsonian folks have leftover reagents, will likely need to order new
will need a reference genome, both for downstream analyses and for adaptive sampling
could also use a de novo genome from sequencing the recent samples
with very fresh tissue, since the genome is p small, could get close to chromosome level genome assembly with nanopore. Andrea notes though that they’ve had a lot of trouble with assembling coral genomes, possibly due to weird genome architecture.
So maybe genome assembly could be a bonus if the species we use ends up being straightforward to assemble, but for now we should try to find samples that at least fall in same genus as a published genome. Will be trickier if we’re using octocorals (for the better sequencing)
Andrea will look at the collections and available octocoral genomes to see if there’s a good candidate.
adaptive sampling could help reduce sequencing of contaminants, but might be less useful with degraded dna (short reads can end up being fully sequenced before pores have a chance to kick them out)
Will ideally want 20x - 30x coverage for methylation calls. Can probably look into what coverage v depth we want for methylation calling
MinION flowcell specs: https://store.nanoporetech.com/us/flow-cell-r10-4-1-2025.html
could try a test run with minion flongle: https://nanoporetech.com/products/sequence/flongle
look/ask around about accessing a promethion.
With the wash kits you don’t actually run the while library each time, you split it up. SO using wash doesn’t require more library.
Will need to check seq length/molarity (Smithsonian has the tools for this)
Expect fragmentation to be an issue. In Andrea’s experience, coral hDNA fragments are max ~1000bp. Should look into how fragmentation could/will affect methylation calling. Could think about a size selection? Should ask folks in meeting next week about their experience
make a list of things Andrea needs to buy.
My to-dos:
Look more into what coverage v depth we want for methylation calling
Look into how use of fragmented DNA can affect methylation calling, including any differences between MinION and PromethION
Looks into accessing a PromethION sequencer at a nearby facility
Johns Hopkins (Baltimore, MD) has a PromethION, but I can’t find additional details for requesting use right now, because the webpage for their Genetic Resources Core Facility is down
NIH Center for Cancer Research (Bethesda, MD) is close , but it looks like they only allow NIH employees/affiliates to use their sequencing facilities. I’d also guess they’re in a lot of tummult right now
UW has a Nanopore Sequencing Core which uses a PromethION. Note sure if I can request a quote from them without sample details (e.g. extraction method, dna concentration), but they have a table of cost estimates here.
If I’m understanding the “services” correctly, it looks like it’s $1,210/flow cell and ~$40/sample for Barcoding with Rapid Prep libraries. I may also be cheaper if we want to multiplex 4 or more samples on a single flow cell.So say we wanted to do 12 samples, multiplex w 4 samples per flow cell: 1210*(3 flow cells) + 40*(12 samples) = $4,110
Optimistically I think we could do it on just two PromethION flow cells: 1210*2+40*12 = $2,900