Gregg Randolph Linda van Diepen Alex Buerkle
This experiment is being repeated due to some errors that occurred during the initial experiment. Experiment protocol below is not finalized.
Questions:
How can we best accommodate low concentration input DNAs?
Concentrate input DNAs?
Concentrate/normalize low yield products?
Both?
Just put more volume of original DNA in?
In the sequence yield, does input concentration, substrate, or some other factor correlate well with low read counts?
...
Pull aliquots of some high read count and low read count samples. (Pending new low read samples).
Quantify all samples not including blanks or mock community.
For previously low yielding samples, repeat at typical template amount, and at 2x, and 4x volume added to the reaction (in column 2,3,4, and 6, 7, 8). 6x8 samples in low yield, 8 in high yield, 8 of mock community, 8 of blank (ISD only). 72 reactions with different templates. See template below for clarification.
Template layout is replicated/duplicated, with different barcodes for duplicates.
1 set of 36 products will not be adjusted for pooling.
1 set of 36 will be adjusted for pooling per qPCR results.
Start Reaction Plate Setup:
...
Sample | Status | Plate | Location | Original Plate Name | DNA Quant ng/uL (>10ng/uL will be normalized) |
|---|---|---|---|---|---|
| Low Read | PA13 | G6 | AYAYEE_RHIZO_PLATE2 | 4.166 |
| Low Read | PA13 | B9 | AYAYEE_RHIZO_PLATE2 | 9.122 |
| Low Read | PA13 | B2 | AYAYEE_RHIZO_PLATE2 | 3.69 |
| Low Read | Tube 33 | 29.422 | ||
| Low Read | Tube 123 | 42.604 | ||
| Low Read | Tube 96 | 2.469 | ||
| Low Read | Tube 258 | 12.88 | ||
| Low Read | Tube 255 | 34.242 | ||
| Avg Read | PA2 | F9 | Tube 127 | 8.333 |
| Avg Read | PA1 | A12 | Tube 73 | 32.279 |
| Avg Read | PA12 | G3 | AYAYEE_RHIZO_PLATE1 | 2.885 |
| Avg Read | PA12 | F7 | AYAYEE_RHIZO_PLATE1 | 6.397 |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|---|---|---|---|---|---|---|---|---|---|
A pool 2 µL each |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only |
B pool 2 µL each |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only |
C pool 2 µL each |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only |
D pool 2 µL each |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
E pool according to yield |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only |
F pool according to yield |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only |
G pool according to yield |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only |
H pool according to yield |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
...
Red samples (bottom half) will either just be pooled per qPCR numbers or concentrated via SpeedVac, reconstituted to a higher concentration, and pooled by qPCR results. (Pending method instructions).
qPCR blue/red pools:
Make 1:1000 dilutions of blue and red pools by adding 1 ul to 999 ul TE in a 1.5mL tubes.
Run each pool in triplicate.
Add 16 ul of Illumina Library Quantification MasterMix to each well:
...