...
...
...
...
...
...
...
...
...
| Info |
|---|
16S0C4 and 16S0D4 were used. |
Gregg Randolph Linda van Diepen Alex Buerkle
This experiment is being repeated due to some errors that occurred in processing during the initial platesexperiment.
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. (New Pending new low read samples requested).
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.
Use ISD at lower than typical concentrations for all for PCRs, as a positive internal control and recognizing that this will wreck reconstruction of absolute counts. Will add ISD to master mix.
Treat templates with previously high read counts in standard manner, except for lower concentration ISD.
Start Reaction Plate Setup:
...
NOTE: Water in the master mix has been adjusted to a lower amount. The plate set up below will replace the water usually added to the master mix while also observing showing if a decrease in water and an increase in template will increase PCR yields.
...
Sample | Status | Plate | Location | Original Plate Name | Low Read | Low Read | Low Read | Low ReadDNA Quant ng/uL (>10ng/uL will be normalized) |
|---|---|---|---|---|---|---|---|---|
| Low ReadLow Read | PA13 | Low Read | Low Read | ||||
| Avg Read | PA2 | F9 | tube | ||||
| Avg Read | PA1 | A12 | tube | ||||
| Avg Read | PA12 | G3G6 | AYAYEE_RHIZO_PLATE2 | 4.166 | |||
| Low Read | PA13 | B9 | AYAYEE_RHIZO_PLATE1PLATE2 | 9.122 | |||
| Low Read | PA12PA13 | F7B2 | AYAYEE_RHIZO_PLATE1 |
1
2
3
4
5
6
7
8
9
A
pool 2 µL each
SAG192203
Tube 127
2ul SAG_S43P4R
PLT2: E11
4ul SAG_S43P4R
PLT2: E11
8ul SAG_S43P4R
PLT2: E11
mock community
2ul SAG_S13P3R
PLT2: A1
4ul SAG_S13P3R
PLT2: A1
8ul SAG_S13P3R
PLT2: A1
blank with ISD only
B
pool 2 µL each
SAG191508
Tube 73
2ul SAG_S10P4R
PLT1: H2
4ul SAG_S10P4R
PLT1: H2
8ul SAG_S10P4R
PLT1: H2
mock community
2ul SAG_S3P2R
PLT1: B1
4ul SAG_S3P2R
PLT1: B1
8ul SAG_S3P2R
PLT1: B1
blank with ISD only
C
pool 2 µL each
SAG_S16P3R
PLT1: G3
2ul SAG_S14P3R
PLT2: E1
4ul SAG_S14P3R
PLT2: E1
8ul SAG_S14P3R
PLT2: E1
mock community
2ul SAG190267
Tube 259
4ul SAG190267
Tube 259
8ul SAG190267
Tube 259
blank with ISD only
D
pool 2 µL each
SAG_S44P2R
PLT1: F7
2ul SAG191507
Tube 74
4ul SAG191507
Tube 74
8ul SAG191507
Tube 74
mock community
2ul SAG190430
Tube 10
4ul SAG190430
Tube 10
8ul SAG190430
Tube 10
blank with ISD only
E
pool according to yield
SAG192203
Tube 127
2ul SAG_S43P4R
PLT2: E11
4ul SAG_S43P4R
PLT2: E11
8ul SAG_S43P4R
PLT2: E11
mock community
2ul SAG_S13P3R
PLT2: A1
4ul SAG_S13P3R
PLT2: A1
8ul SAG_S13P3R
PLT2: A1
blank with ISD only
F
pool according to yield
SAG191508
Tube 73
2ul SAG_S10P4R
PLT1: H2
4ul SAG_S10P4R
PLT1: H2
8ul SAG_S10P4R
PLT1: H2
mock community
2ul SAG_S3P2R
PLT1: B1
4ul SAG_S3P2R
PLT1: B1
8ul SAG_S3P2R
PLT1: B1
blank with ISD only
G
pool according to yield
SAG_S16P3R
PLT1: G3
2ul SAG_S14P3R
PLT2: E1
4ul SAG_S14P3R
PLT2: E1
8ul SAG_S14P3R.
PLT2: E1
mock community
2ul SAG190267
Tube 259
4ul SAG190267
Tube 259
8ul SAG190267
Tube 259
blank with ISD only
H
pool according to yield
SAG_S44P2R
PLT1: F7
2ul SAG191507
Tube 74
4ul SAG191507
Tube 74
8ul SAG191507
Tube 74
mock community
2ul SAG190430
Tube 10
4ul SAG190430
Tube 10
8ul SAG190430
Tube 10
blank with ISD only
Add 2 ul 1-step primers:
Seal with bubble seals. Vortex briefly. Spin down.
Run on Thermocycler Program GSAF36:
...
Temp C
...
Cycles
...
Time
...
95*
...
1X
...
3:00*
...
98
...
36X
...
0:30
...
62
...
36X
...
0:30
...
72
...
36X
...
0:30
...
72
...
1X
...
5:00
...
4
...
1X
...
0:00
MagBead Cleanup:
Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”
Manually, it was done:
Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)
qPCR all products:
Make 1:1000 dilutions of all samples from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
...
...
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
12
...
A
...
blank with ISD only
...
...
...
NTC
...
B
...
blank with ISD only
...
...
04.0002 pM Std
...
0.0002 pM Std
...
C
...
blank with ISD only
...
...
0.002 pM Std
...
0.002 pM Std
...
D
...
blank with ISD only
...
...
0.02 pM Std
...
0.02 pM Std
...
E
...
blank with ISD only
...
...
0.2 pM Std
...
0.2 pM Std
...
F
...
blank with ISD only
...
...
2 pM Std
...
2 pM Std
...
G
...
blank with ISD only
...
...
20 pM Std
...
20 pM Std
...
H
...
blank with ISD only
...
...
...
Add 16 ul of Illumina Library Quantification MasterMix to each well:
...
ul/rxn
...
Reagent
...
# of rxns
...
ul needed
...
10 ul
...
KAPA SYBR FAST qPCR MM (2X)
...
100
...
1000
...
2 ul
...
Primer Premix (10X)
...
100
...
200
...
4 ul
...
Ultra Pure Water
...
100
...
400
...
16 ul
...
Total Volume
...
100
...
1600
Add 4 ul of template, pool, or standards to each well:
...
...
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
12
...
A
...
SAG192203
Tube 127
...
2ul SAG_S43P4R
PLT2: E11
...
4ul SAG_S43P4R
PLT2: E11
...
8ul SAG_S43P4R
PLT2: E11
...
mock community
...
2ul SAG_S13P3R
PLT2: A1
...
4ul SAG_S13P3R
PLT2: A1
...
8ul SAG_S13P3R
PLT2: A1
...
blank with ISD only
...
NTC
...
NTC
...
NTC
...
B
...
SAG191508
Tube 73
...
2ul SAG_S10P4R
PLT1: H2
...
4ul SAG_S10P4R
PLT1: H2
...
8ul SAG_S10P4R
PLT1: H2
...
mock community
...
2ul SAG_S3P2R
PLT1: B1
...
4ul SAG_S3P2R
PLT1: B1
...
8ul SAG_S3P2R
PLT1: B1
...
blank with ISD only
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
SAG_S16P3R
PLT1: G3
...
2ul SAG_S14P3R
PLT2: E1
...
4ul SAG_S14P3R
PLT2: E1
...
8ul SAG_S14P3R
PLT2: E1
...
mock community
...
2ul SAG190267
Tube 259
...
4ul SAG190267
Tube 259
...
8ul SAG190267
Tube 259
...
blank with ISD only
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
SAG_S44P2R
PLT1: F7
...
2ul SAG191507
Tube 74
...
4ul SAG191507
Tube 74
...
8ul SAG191507
Tube 74
...
mock community
...
2ul SAG190430
Tube 10
...
4ul SAG190430
Tube 10
...
8ul SAG190430
Tube 10
...
blank with ISD only
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
SAG192203
Tube 127
...
2ul SAG_S43P4R
PLT2: E11
...
4ul SAG_S43P4R
PLT2: E11
...
8ul SAG_S43P4R
PLT2: E11
...
mock community
...
2ul SAG_S13P3R
PLT2: A1
...
4ul SAG_S13P3R
PLT2: A1
...
8ul SAG_S13P3R
PLT2: A1
...
blank with ISD only
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
SAG191508
Tube 73
...
2ul SAG_S10P4R
PLT1: H2
...
4ul SAG_S10P4R
PLT1: H2
...
8ul SAG_S10P4R
PLT1: H2
...
mock community
...
2ul SAG_S3P2R
PLT1: B1
...
4ul SAG_S3P2R
PLT1: B1
...
8ul SAG_S3P2R
PLT1: B1
...
blank with ISD only
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
SAG_S16P3R
PLT1: G3
...
2ul SAG_S14P3R
PLT2: E1
...
4ul SAG_S14P3R
PLT2: E1
...
8ul SAG_S14P3R.
PLT2: E1
...
mock community
...
2ul SAG190267
Tube 259
...
4ul SAG190267
Tube 259
...
8ul SAG190267
Tube 259
...
blank with ISD only
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
SAG_S44P2R
PLT1: F7
...
2ul SAG191507
Tube 74
...
4ul SAG191507
Tube 74
...
8ul SAG191507
Tube 74
...
mock community
...
2ul SAG190430
Tube 10
...
4ul SAG190430
Tube 10
...
8ul SAG190430
Tube 10
...
blank with ISD only
...
...
...
Results:
Pool all samples:
Blue samples (top half of plate) will be pooled at 1* ul per sample.
*The original experimental design was to add 2uL per sample for the blue pool but with the extremely low yields occurring in the red half of the plate I reduced the pooling amount of the blue pool to make the two pools closer in total concentration.
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.
NOTE: Row H showed significantly lower yields. I believe this could have occurred due to evaporation of the PCR master mix. The master mix was prepared ahead of time, plated, and refrigerated until samples could be loaded. We prepare master mix plates ahead of time often, with no noticeable evaporation, but I did notice a slight decrease in volume on the outer edges of the plate during sample loading. Due to the master mix being specialized for this experiment, I proceeded with the plates as is since they were in duplicate. If the following steps below do not show the desired result, we could repeat the experiment with the master mix preparation and sample loading occurring on the same day.
Calculations for Red Pool:
Since the majority of the samples in the red pool were in the 10-99 nanomole range, the following samples will be added in higher quantities to ensure the samples are pooled in the same order of magnitude:
In theory, if you decrease the elution volume by 50%, the DNA concentration should increase by 50%. That being said, in order for some of the samples to reach the desired concentration, we will need to vacuum concentrate and resuspend at a lower elution volume.
samples with concentrations between 10-99 will have 1uL added to the pool.
samples highlighted in red were too low to normalize to 10 nanomoles so I just resuspended them at 10uL and used half to at least increase the input quantity.
...
Sample
...
qPCR Result
(nanomoles)
...
Quantity in sample (39uL)
(nanomoles)
...
Elution volume post SpeedVac
(uL)
...
Quantity post SpeedVac
(nanomoles)
...
Amount to add to pool
(uL)
...
Quantity in final pool
(nanomoles)
...
SAG192203_R (E1)
...
0.273
...
10.65
...
10
...
41.52
...
2.4
...
10
...
SAG_S43P4R_R (E4)
...
0.0411
...
1.60
...
4
...
15.36
...
2.6
...
10
...
SAG_S13P3R_R (E8)
...
0.000475
...
0.02
...
10
...
0.08
...
5
...
0.04
...
BLANK_ISD (E9)
...
0.215
...
8.39
...
10
...
32.71
...
3.1
...
10
...
SAG191508_R (F1)
...
1.58
...
61.62
...
...
...
6.3
...
10
...
SAG_S10P4R_R (F4)
...
0.291
...
11.35
...
10
...
44.25
...
2.3
...
10
...
SAG_S3P2R_R (F8)
...
0.673
...
26.25
...
20
...
51.17
...
3.9
...
10
...
BLANK_ISD (F9)
...
0.445
...
17.36
...
20
...
33.84
...
5.9
...
10
...
SAG_S14P3R_R (G3)
...
0.218
...
8.50
...
10
...
33.14
...
3.0
...
10
...
SAG_S14P3R_R (G4)
...
0.00121
...
0.05
...
10
...
.19
...
5
...
0.095
...
SAG190267_R (G8)
...
0.0154
...
0.60
...
4
...
5.76
...
2
...
2.88
...
SAG191507_R (H3)
...
0.000115
...
0.004
...
10
...
.02
...
5
...
0.01
...
SAG191507_R (H4)
...
0.00247
...
0.10
...
10
...
0.39
...
5
...
0.195
...
Mock_Comm_R (H5)
...
0.148
...
5.77
...
10
...
22.50
...
4.4
...
10
...
SAG190430_R (H6)
...
0.127
...
4.95
...
10
...
19.30
...
5.2
...
10
...
SAG190430_R (H7)
...
0.00073
...
0.03
...
10
...
.12
...
5
...
0.06
...
SAG190430_R (H8)
...
0.000326
...
0.01
...
10
...
.04
...
5
...
0.02
...
BLANK_ISD (H9)
...
0.00311
...
0.12
...
10
...
.47
...
5
...
0.235
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:
...
ul/rxn
...
Reagent
...
# of rxns
...
ul needed
...
10 ul
...
KAPA SYBR FAST qPCR MM (2X)
...
30
...
300
...
2 ul
...
Primer Premix (10X)
...
30
...
60
...
4 ul
...
Ultra Pure Water
...
30
...
120
...
16 ul
...
Total Volume
...
30
...
480
Add 4 ul of template, pool, or standards to each well:
1
2
3
4
5
6
7
8
9
10
11
12
A
LR_Blue_Pool
NTC
NTC
NTC
B
LR_Blue_Pool
0.0002 pM Std
0.0002 pM Std
0.0002 pM Std
C
LR_Blue_Pool
0.002 pM Std
0.002 pM Std
0.002 pM Std
D
LR_Red_Pool
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 |
Add 2 ul 1-step primers:
Seal with bubble seals. Vortex briefly. Spin down.
Run on Thermocycler Program GSAF36:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00* |
98 | 36X | 0:30 |
62 | 36X | 0:30 |
72 | 36X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
MagBead Cleanup:
Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”
Manually, it was done:
Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)
qPCR all products:
Make 1:1000 dilutions of all samples from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only |
|
| NTC |
B |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only |
| 04.0002 pM Std | 0.0002 pM Std |
C |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only |
| 0.002 pM Std | 0.002 pM Std |
D |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
| 0.02 pM Std | 0.02 pM Std |
E |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only |
| 0.2 pM Std | 0.2 pM Std |
F |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only |
| 2 pM Std | 2 pM Std |
G |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only |
| 20 pM Std | 20 pM Std |
H |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
|
|
|
Add 16 ul of Illumina Library Quantification MasterMix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 100 | 1000 |
2 ul | Primer Premix (10X) | 100 | 200 |
4 ul | Ultra Pure Water | 100 | 400 |
16 ul | Total Volume | 100 | 1600 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only | NTC | NTC | NTC |
B |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only | 2 pM Std | 2 pM Std | 2 pM Std |
G |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only | 20 pM Std | 20 pM Std | 20 pM Std |
H |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
|
|
|
Results:
| View file | ||
|---|---|---|
|
Pool all samples:
Blue samples (top half of plate) will be pooled at 1* ul per sample.
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).
“ul for pooling” was calculated by multiplying the lowest molarity (3.15) times the maximum V available (35 ul) to get ~110. This result was then divided by each sample’s molarity to obtain the ul needed for equimolar pooling rounded to one decimal place.
This will need to be done by hand, but if we pursue this I will tweak the Nimbus normalization program to do this. For a NovaSeq run, I would pick a higher minimum molarity. While this pool should be above the NovaSeq minimum molarity needed of 1 nM, being higher than its 3.15 nM minimal constituent, I would prefer a larger margin of safety like 5 nM. For an iSeq run, we dilute down to 50 pM, so it is not an issue.
Well Position | Sample | qPCR result | ul for pooling | Repeated qPCR Result | qPCR Duplicated Run Results 02/09 |
1E | SAG192203_R | 54.32 | 2.0 | 1.58 | 2.04 |
2E | SAG_S30P2R_R_2uL | 54.14 | 2.0 | 1.22 | 1.16 |
3E | SAG_S30P2R_R_4uL | 54.03 | 2.0 | 2.05 | 1.34 |
4E | SAG_S30P2R_R_8uL | 11.34 | 9.7 | 5.70E-01 | 3.61E-01 |
5E | Mock_Comm_R | 65.14 | 1.7 | 2.07 | 1.62 |
6E | SAG192199_R_2uL | 71.08 | 1.5 | 2.09 | 1.25 |
7E | SAG192199_R_4uL | 95.41 | 1.2 | 2.34 | 3.91 |
8E | SAG192199_R_8uL | 80.56 | 1.4 | 2.65 | 6.07 |
9E | Blank_ISD_R | 19.23 | 5.7 | 1.8 | 2.25 |
1F | SAG191508_R | 64.52 | 1.7 | 31.69 | 37.26 |
2F | SAG_S38P1R_R_2uL | 60.01 | 1.8 | 1.51 | 1.56 |
3F | SAG_S38P1R_R_4uL | 56.04 | 2.0 | 6.72 | 5.75 |
4F | SAG_S38P1R_R_8uL | 3.15 | 34.9 | 3.86E-01 | 1.42E-01 |
5F | Mock_Comm_R | 63.52 | 1.7 | 2.51 | 2.11 |
6F | SAG192077_R_2uL | 65.88 | 1.7 | 2.38 | 1.58 |
7F | SAG192077_R_4uL | 84.27 | 1.3 | 2.37 | 3.1 |
8F | SAG192077_R_8uL | 83.59 | 1.3 | 3.03 | 4.24 |
9F | Blank_ISD_R | 13.12 | 8.4 | 7.78E-01 | 9.15E-01 |
1G | SAG_S16P3R_R | 26.49 | 4.2 | 11.34 | 14.1 |
2G | SAG_S15P4R_R_2uL | 53.51 | 2.1 | 14.31 | 16.26 |
3G | SAG_S15P4R_R_4uL | 26.2 | 4.2 | 1.33 | 1.56 |
4G | SAG_S15P4R_R_8uL | 12.91 | 8.5 | 6.56E-01 | 6.53E-01 |
5G | Mock_Comm_R | 79.7 | 1.4 | 1.9 | 2.14 |
6G | SAG190271_R_2uL | 70.2 | 1.6 | 1.59 | 1.75 |
7G | SAG190271_R_4uL | 62.73 | 1.8 | 1.81 | 1.95 |
8G | SAG190271_R_8uL | 59.32 | 1.9 | 6.33E-01 | 2.61 |
9G | Blank_ISD_R | 20.04 | 5.5 | 6.79E-01 | 1.13 |
1H | SAG_S44P2R_R | 19.92 | 5.5 | 2.4 | 4.46 |
2H | SAG190005_R_2uL | 54.03 | 2.0 | 2.89 | 6.26 |
3H | SAG190005_R_4uL | 57.05 | 1.9 | 2.72 | 3.62 |
4H | SAG190005_R_8uL | 60.28 | 1.8 | 4.37 | 3.46 |
5H | Mock_Comm_R | 64.83 | 1.7 | 2.17 | 2.28 |
6H | SAG190238_R_2uL | 68.19 | 1.6 | 76.62 | 62 |
7H | SAG190238_R_4uL | 55.07 | 2.0 | 2.16 | 1.83 |
8H | SAG190238_R_8uL | 41.35 | 2.7 | 2.5 | 3.88 |
9H | Blank_ISD_R | 18.32 | 6.0 | 5.57E-01 | 1.36 |
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:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 30 | 300 |
2 ul | Primer Premix (10X) | 30 | 60 |
4 ul | Ultra Pure Water | 30 | 120 |
16 ul | Total Volume | 30 | 480 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | LR2_Blue_Pool |
|
|
|
|
|
|
|
| NTC | NTC | NTC |
B | LR2_Blue_Pool |
|
|
|
|
|
|
|
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | LR2_Blue_Pool |
|
|
|
|
|
|
|
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | LR2_Red_Pool |
|
|
|
|
|
|
|
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | LR2_Red_Pool |
|
|
|
|
|
|
|
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | LR2_Red_Pool |
|
|
|
|
|
|
|
| 2 pM Std | 2 pM Std | 2 pM Std |
G |
|
|
|
|
|
|
|
|
| 20 pM Std | 20 pM Std | 20 pM Std |
H |
|
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|
|
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|
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|
Results:
Average Results:
LR2_Blue_Pool: 53.65 nanomoles
LR2_Red_Pool: 35.33 nanomoles
| View file | ||
|---|---|---|
|
iSeq Run
Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.
Blue Pool
1000/Results = ul of Pool to Add
1000/53.7 = 18.6 uL of Pool to Add
1000- ul of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000- 18.6 = 981.4 uL of 10mM Tris 8.5
Red Pool
1000/Results = ul of Pool to Add
1000/35.3 = 28.3 uL of Pool to Add
1000- ul of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000- 28.3= 971.7 uL of 10mM Tris 8.5
Combine Blue and Red pools in equal parts once pools are at 1nM
Dilute 1 nM full pool to loading concentration of 50 pM:
Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.
Results located: Data/SequencingRuns/”folder with applicable date”/Alignment_1/Fastq/*.fastq.gz
Upload data through Globus portal off external drive
Data wrangling(2):
Use R to associate NS2 input concentrations with sample names, read counts, and % ISD reads
Repeat qPCR post calibration:
qPCR MMX
Add 16 ul of Illumina Library Quantification MasterMix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 100 | 1000 |
2 ul | Primer Premix (10X) | 100 | 200 |
4 ul | Ultra Pure Water | 100 | 400 |
16 ul | Total Volume | 100 | 1600 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only | NTC | NTC | NTC |
B |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E |
LR_Red_Pool
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |
F |
LR_Red_Pool
2 pM Std
2 pM Std
2 pM Std
G
20 pM Std
20 pM Std
20 pM Std
H
Results:
Average Results:
LR_Blue_Pool: 18.10 nanomoles
LR_Red_Pool: 8.36 nanomoles
iSeq Run
Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.
*Due to the blue pool only having a total of 36uL, the 1nM pool calculations had to be at a total volume of 500uL instead of 1000uL.
Blue Pool
500/Results = ul of Pool to Add
500/18.1= 27.5 uL of Pool to Add
500- ul of Pool to Add = ul of “10 mM Tris 8.5” to Add
500- 27.5 = 472.5 uL of 10mM Tris 8.5
Red Pool
500/Results = ul of Pool to Add
500/8.36 = 59.8 uL of Pool to Add
500- ul of Pool to Add = ul of “10 mM Tris 8.5” to Add
500- 59.8= 440.2 uL of 10mM Tris 8.5
Combine Blue and Red pools in equal parts once pools are at 1nM
Dilute 1 nM full pool to loading concentration of 50 pM:
Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.
Results located: Data/SequencingRuns/”folder with applicable date”/Alignment_1/Fastq/*.fastq.gz
Data wrangling(2):
...
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only | 2 pM Std | 2 pM Std | 2 pM Std | |
G |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only | 20 pM Std | 20 pM Std | 20 pM Std |
H |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
|
|
|
Results 02/08/2022:
| View file | ||
|---|---|---|
|
Results for duplicate qPCR run on 02/09/2022:
| View file | ||
|---|---|---|
|
Results look the same from the 02/08 run.