NS5 Contamination inquiry continued
- Gregg Randolph
12-09-2022 Most likely culprit is primers.
I examined the zotus_nonchimeric.fa files for each primer plate; the demux key assigned a different project to each primer source. I looked at the first 5-7 most abundant otus from each. The two fresh primer plates were consistent and the difference was with the original primer plate.
Inquiry
Sources of Contamination to Test:
Normalization/TE
Do new normalization of 5FB1
Repeat 16S with original primers and primers not used with Alfalfa
MasterMix
Tested and likely not sole culprit if culprit at all
Medicago still shows up
Primers
Repeat 16S of 5FB1 again with primers not used with Alfalfa
Beads
Process NS5 with barcode pairs that should not be present
Master Mixes used with links to QC pages
PCR
Pull Out 5FB1_Norm and 5FB1 and allow to thaw; wash hands before proceeding.
Renormalize 5FB1 label 5FB1_Renorm1
Pull Out 3 hard shell, full skirt plates and label them NS5FB1_16S0G11, NS5FB1_16S0C4, and NS5FB1_16S0H11
Pull Out 10-20-22_AmpliconMM_2 and allow to thaw
Pull Out long16S0C4, long16S0G11, and long16S0H11 and vortex and spin down
Add 11 ul MM to each well of the 3 hard shell, full skirt plates. Save MM remainder in a clean 2 ml tube. Transfer label.
Add 2 ul 5FB1_Norm to NS5FB1_16S0G11
Add 2 ul 5FB1_Renorm1 to NS5FB1_16S0C4, and NS5FB1_16S0H11
Add 2 ul from the appropriate primer plate to each plate. Seal, vortex, and spin.
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 |
Remember to save any remaining MM in a 2 ml tube, transfer label from 5 ml, and return to -20 for possible future retesting.
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)
Store in -20 until ready to qPCR
qPCR NovaSeq5_ConTest1
Make 1:1000 dilutions of column 8 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 |
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| NTC | NTC | NTC |
B |
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| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C |
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| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D |
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| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E |
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| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F |
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| 2 pM Std | 2 pM Std | 2 pM Std |
G |
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| 20 pM Std | 20 pM Std | 20 pM Std |
H |
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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) | 110 | 1100 |
2 ul | Primer Premix (10X) | 110 | 220 |
4 ul | Ultra Pure Water | 110 | 440 |
16 ul | Total Volume | 110 | 1760 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
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| 20 pM Std | 20 pM Std | 20 pM Std |
B |
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| 2 pM Std | 2 pM Std | 2 pM Std |
C |
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| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
D |
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| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E |
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| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
F |
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| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
G |
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| NTC | NTC | NTC |
H |
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| NTC | NTC | NTC |
Results:
Plate Name | Average Result (nanomoles) |
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Full qPCR results below:
Sequencing Test:
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.
1000/Results = ul of Pool to Add
100/8.4 = 12uL of Pool to Add
100 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
100- 12 = 82 uL of 10mM Tris 8.5
Dilute 1 nM full pool to loading concentration of 60 pM:
Add 6 ul 1 nM Pool to 82 ul “10 mM Tris 8.5” and 12 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.
This run failed without detecting any clusters. So, we are repeating it.
Redone Sequencing Test:
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.
1000/Results = ul of Pool to Add
100/10 = 10uL of Pool to Add
100 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
100- 10 = 90 uL of 10mM Tris 8.5
Dilute 1 nM full pool to loading concentration of 60 pM:
Add 6 ul 1 nM Pool to 82 ul “10 mM Tris 8.5” and 12 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.