Post date: Apr 18, 2018 10:6:28 PM
Here are a few key notes related to my correspondence with Lucigen about the genome.
Here is my description of what we sent:
"I included five vials, each with about 20 beetles (mostly adults, a few pupae and larvae). Samples within each jar are siblings from inbred lines. All of the lines are quite inbred, with inbreeding coefficients of 0.786 (2 tubes), 0.827 (2 tubes) and 0.86 (1 tube) (higher values indicate additional generations of inbreeding). I would like the fragment library to come from a single tube (inbred line), and if possible this would be true of the all or as many libraries as possible. Note that tubes with the same color sticker on top are actually the same line at different generations, so if tubes must be combined to get enough DNA, combining tubes with the same stickers is the best bet. I also included the signed contract with the shipment."
Note that those should denote 7, 8 and 9 generations of full-sib mating, respectively.
And here are some notes on what they did (from Brendan):
1. "Long story short: extracted from one red dot vial (0.827 inbreeding coefficient), obtained ~ 60 micrograms all told, looks RNA free and HMW – should be good to go...
More detail: the RNA check gel showed some smearing to small MWs. After polling the team (and our mate pair development expert), we decided that these could cause a problem if we let them hang around, so we won’t. Ordinarily, we’d proceed to shearing, then a quick SPRI cleanup/concentration, then modification & adaptors and so on through the process, but in this case that SPRI cleanup will be more stringent and play more like a size-selection against the small stuff. I should be able to finish that up before the holiday at least for the frag, 3 kb, and 8 kb. This will tell us where we stand and if additional red dot sample extraction is necessary – I’m hoping we can do this project with what’s on hand (as you stated a preference for that), but losses are expected to be higher as insert size increases."
2. "Quick update. A single vial’s extract covered the fragment, 3kb, and 8 kb libraries. We may have to pool the two red-dot extracts for the 20 kb – but this is in progress and we’ll need a few days to reach the critical QC checkpoint. I do have an extract from one of the yellow dot backups, just in case. It may be that we need to pool some of that too, but again, we need a few days to see if this is necessary. The extracts have been consistently 20-100 kb in size plus some of the smearing to small MWs as noted previously; if it turns out we don’t use all of it (probable), would you like me to send you the spare extracts? That is, if you have a use for them…"
3. "Another update:
1. Frag, 3kb, and 8 kb all were constructed from the red dot vial with inbreeding coefficient of 0.827, and are ready to sequence.
2. The yield wasn’t quite enough to support the 20 kb also, and required extracting the second red vial (0.786). My tech was unable to recover enough, though she did try to pool what material she had.
3. I then extracted from yellow dot (0.86) and 20 kb is underway.
I am sorry about that – I was hoping to get it all from the one vial. Regarding the sequencing for the fragment, I’d like to tie up your project before the end of the year, but I note you have high output mode selected (10 days). According to our UW collaborators with the HiSeq 2500, the Rapid mode will also produce on average 150M reads per lane (they usually get more) in three days time, and I think they encourage that since it helps them wade through the huge sequencing volume they get without penalty to the customer for the overall amount of data generated. The prices are virtually indistinguishable: https://www.biotech.wisc.edu/services/dnaseq/sequencing/Illumina - and we have it quoted at a slight loss.
What I wanted to know is whether or not you would like to switch modes at the same read length – it may well be that the holdup for us would be that the cores typically don’t run the rarer requests until they can fill up the flowcell, and their indication to us is that rapid is more common for this read length. Either way, I leave it up to you."