AmandaBirmingham
commented
5 years ago Amanda's notes from miniPCR protocol discussion re LabControl Charlie, MacKenzie, Amanda, Gail 04/12/2019
MiniPCR: kind of combination of 16s and shotgun
plate samples individually in 96well plates just as with amplicon
extract dna just as with amplicon
plate consolidation--same as shotgun protocol (compress gdna plates)
PCR: need to do prepare amplicon libraries but with a compressed plate (384 plate)
Amplicon library prep
need to have primers--primer sets are compressed just like the regular plates (just two primer sets)
primer set 1 is EMP 16s 1-4, primer set 2 is 5-8
don't use epmotion for actual transfer of gdna and primer, do use if for dispensing master mix; for this, do NOT use TM300, just TM50
need to indicate that transfer is on the echo ... but maybe that's implicit
Would be great to have a notes field so can record any issues with the echo--and that applies to the entire process, not just one plate
for minipcr, majority of projects MacKenzie has done have NOT used pico green quantification, have just gone to quantifying at equal pooling. So need to be able to ignore this step and go straight to pooling, but also need the option to do that step if we want to.
SO since not doing quantification and pooling everything at fixed volume, don't need a new pooling file from LabControl every time (still need a pooling file, but it is the same one every time--just says pool each of the 384 wells on the plate at 2 microLiters, and file doesn't need sample info).
If WERE doing quantification (on 384-well library prep plate) on "Pool library plate", total quantity of dna to pool per sample would be 40 ng. If required to pool more than 10 ml, will just pool ten instead (so if concentration is super low and it wants us to pool much more, we will still only pull 10 bc don't want to over dilute anything.) Also a lower limit--if the volume to be pooled is under 1 ml, will bump up to 1 ml. <--note: don't think have a lower limit for the existing amplicon pooling.
So far besides basic emp testing, have always done equal volume testing.
MacKenzie: I usually think in volumes, so the "minimum value for pooling at real estimated value (ng/uL)" which is in concentrations requires a bit of mental calculations to figure out what to put in.
Trying to pool 6 96-plates into one pool for a run on MiSeq (that works well for us) but that is 1.5 96-well plates, so part of a plate will go to one run, part will go to another run. Want to pick which "half" of the 384 well plate goes into the pool based on which 96-well plates that half came from.
For 16s normally, would quantify each pooled plate tube before combining; that gives us quantifications to understand how much we have to pool together. But since I'm combining everything before, quantify at the very very end.
For the miniPCR protocol, the pool that is created by Pool Library Plate (that includes 1.5 of a 384-well plate) actually IS the Sequencing Pool. There is one more quantification of that pool done by MacKenzie before loading the MiSeq, but that quantification is used only by the miseq and isn't currently stored in LabControl.
Need to be able to create a sequencing run using the result of Pool Library Plate, not Prepare Sequencing Pool.
Ran through a single plate (HNRC) that was part of a bigger sequencing run, but only uploaded HNRC to LabControl, but didn't run Prepare Amplicon Sequencing Pool. Note: were still offered options to download sample sheet and prep sheet, but they were corrupt.
Can't just run Prepare Sequencing Pool with a single UNQUANTIFIED pool ... could put in dummy concentration value. <--obviously bad to put in dummy values, need to find out where these are used and what repercussions of dumminess are.
charles-cowart
Mini16S protocol is a newer protocol that reduces cost and time. It is not currently supported by LabControl.
This description is a placeholder issue to be fleshed out at a later date.