| Info |
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New aliquots of the 4 pools were mailed to the University of Colorado on 10/13/2021 for deeper sequencing. Data was retrieved on 11/09/2021 The four pools were mailed to the University of Colorado’s Genomics Core on 03/23/2021. Pools were ran on 03/30/21. Data uploaded 4/02/2021. |
Use iProof Polymerase for this. We can play around with KAPA afterwards to see if we don’t need 2 taqs in house
Used this r-script to bind the Masterkey to the pool and mid plate map, this plate map, and the 2017 plate map to make this demux key
Sequencing Reports Nov 2021
Lane Summary (Pools 1 and 2) AHNYYMDRXY
Pool | Lane | PF Clusters | % of the | % Perfect | % One mismatch | Yield (Mbases) | % PF | % >= Q30 | Mean Quality |
|---|---|---|---|---|---|---|---|---|---|
1 | 1 | 963,211,753 | 100 | 100 | NaN | 104,990 | 75.45 | 89.09 | 34.84 |
2 | 2 | 926,591,105 | 100 | 100 | NaN | 100,998 | 72.58 | 89.23 | 34.86 |
Lane Summary (Pools 3 and 4) BHNYW7DRXY
Pool | Lane | PF Clusters | % of the | % Perfect | % One mismatch | Yield (Mbases) | % PF | % >= Q30 | Mean Quality |
|---|---|---|---|---|---|---|---|---|---|
3 | 1 | 972,876,446 | 100 | 100 | NaN | 106,044 | 76.2 | 89.69 | 34.95 |
4 | 2 | 918,555,590 | 100 | 100 | NaN | 100,123 | 71.95 | 88.79 | 34.79 |
Sequencing Reports April 2021
Flowcell Summary (Pools 1 and 2)
Clusters (Raw) | Clusters(PF) | Yield (MBases) |
|---|---|---|
1,276,674,048 | 821,869,647 | 89,584 |
Lane Summary (Pools 1 and 2)
Pool | Lane | PF Clusters | % of the | % Perfect | % One mismatch | Yield (Mbases) | % PF | % >= Q30 | Mean Quality |
|---|---|---|---|---|---|---|---|---|---|
1 | 1 | 416,256,593 | 100.00 | 100.00 | NaN | 45,372 | 65.21 | 86.76 | 34.40 |
2 | 2 | 405,613,054 | 100.00 | 100.00 | NaN | 44,212 | 63.54 | 86.89 | 34.42 |
Flowcell Summary (Pools 3 and 4)
Clusters (Raw) | Clusters(PF) | Yield (MBases) |
|---|---|---|
1,276,674,048 | 909,753,754 | 99,163 |
Lane Summary (Pools 3 and 4)
Pool | Lane | PF Clusters | % of the | % Perfect | % One mismatch | Yield (Mbases) | % PF | % >= Q30 | Mean Quality |
|---|---|---|---|---|---|---|---|---|---|
3 | 1 | 482,619,189 | 100.00 | 100.00 | NaN | 52,605 | 75.61 | 89.39 | 34.89 |
4 | 2 | 427,134,565 | 100.00 | 100.00 | NaN | 46,558 | 66.91 | 87.90 | 34.62 |
Reagents and Ordering
- EcoR1 (20,000 units/ml)
- MseI (10,000 units/ml)
- T4 DNA ligase buffer (400,000 units/ml)
- DNA polymerase (BioRad iProof or KAPA HiFi)
- BSA (1 mg/ml)-We have this in 20 mg/ul
- 1 M NaCl-We have this at 5M
- DMSO
- EcoR1 adaptors (We need 1 uM MIDed in plates)
- Mse1 Adaptors (10 uM stock probably need to dilute from 100 uM)
- IIllpcr2 (10 uM working)
- Illpcr1 (10 uM working)
- Pool of above 2 primers (5 uM working of each)
...
Add 3 ul Digestion MM to 16 plates using the Benchsmart
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 1800 | 2070 |
5M NaCl | 0.12 | 1800 | 216 |
1 mg/ml BSA | 0.6 | 1800 | 1080 |
H2O | 0.73 | 1800 | 1314 |
MseI (enzyme) | 0.12 | 1800 | 216 |
EcoR1 (enzyme) | 0.28 | 1800 | 504 |
Total | 3 | 1800 | 5400 |
Use an 8 channel to divvy up 71 ul across a plate and then the Benchsmart to add 3 ul to 22 plates.
...
Spin down plates and add 1.4 ul Ligation MM to each well with Benchsmart.
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
MseI oligo | 1 | 1800 | 1800 |
H2O | 0.112 | 1800 | 201.6 |
10x T4 Buffer | 0.1 | 1800 | 180 |
5M NaCl | 0.01 | 1800 | 18 |
1 mg/ml BSA | 0.05 | 1800 | 90 |
T4 DNA ligase | 0.1675 | 1800 | 301.5 |
Total | 1.4 | 1800 | 2591.1 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
2017_ALFALFA_PLATE5 | 5 |
2017_ALFALFA_PLATE6 | 6 |
2017_ALFALFA_PLATE7 | 7 |
2017_ALFALFA_PLATE8 | 8 |
2017_ALFALFA_PLATE9 | 1 |
Label reaction plates with MID plate used.
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Pool ligated plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together:
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
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We are running duplicate PCRs for each template.
Add 16 ul of PCR1 MM to each well of plates
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 900 | 8568 |
5x iProof buffer | 4 | 900 | 3600 |
10 mM dNTPs | 0.4 | 900 | 360 |
50 mM MgCl2 | 0.4 | 900 | 360 |
5 uM Illumina Primers | 1.33 | 900 | 1197 |
iProof TAQ | 0.2 | 900 | 180 |
DMSO | 0.15 | 900 | 135 |
total | 16 | 900 | 14400 |
Add 4 ul of restriction/ligation products to each well
Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 30X | 0:30 |
60 | 30X | 0:30 |
72 | 30X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
*pause step
Extra PCR
Add 2.125 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | 900 | 382.5 |
10 mM dNTPs | 0.4 | 900 | 360 |
Primers | 1.33 | 900 | 1197 |
Total | 2.155 | 900 | 1939.5 |
Then continue with the last four unlisted steps for GBS1 from above:
Temp C | Cycles | Time |
|---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Pooling
Sequencing Pool | PCR Pool | PCR Pool |
|---|---|---|
seqPool1 | Pool1 | Pool2 |
seqPool2 | Pool3 | Pool4 |
seqPool3 | Pool5 | Pool6 |
Size Selection
We will utilize utilized the Pippin Prep machine on loan from the Ernest Lab, selecting for the range 300-450bp.