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Info

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

Sequencing Reports Nov 2021

Lane Summary (Pools 1 and 2) AHNYYMDRXY

Pool

Lane

PF Clusters

% of the
lane

% Perfect
barcode

% One mismatch
barcode

Yield (Mbases)

% PF
Clusters

% >= Q30
bases

Mean Quality
Score

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
lane

% Perfect
barcode

% One mismatch
barcode

Yield (Mbases)

% PF
Clusters

% >= Q30
bases

Mean Quality
Score

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
lane

% Perfect
barcode

% One mismatch
barcode

Yield (Mbases)

% PF
Clusters

% >= Q30
bases

Mean Quality
Score

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
lane

% Perfect
barcode

% One mismatch
barcode

Yield (Mbases)

% PF
Clusters

% >= Q30
bases

Mean Quality
Score

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)

...

Normalize to between 20 ng/ul and 150 ng/ul

2017 alfalfa: Transfer to conical pcr plates. Normalize on nimbus to 100 ng/ul in 30 ul.

Illumina Primer Pooling

Add 900 ul std TE to 5 tubes

...

Only needs to be done after first making working stock.

Add 1200 ul std TE to three two 2 ml tubes.

Add 150 ul MseI1 to each tube

...

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 20 16 plates using the Benchsmart

Reagent

ul/rxn

rxns

ul needed

10x T4 Buffer

1.15

2220

1800

2553

2070

5M NaCl

0.12

2220

1800

266.4

216

1 mg/ml BSA

0.6

2220

1800

1332

1080

H2O

0.73

2220

1800

1620.6

1314

MseI (enzyme)

0.12

2220

1800

266.4

216

EcoR1 (enzyme)

0.28

2220

1800

621.6

504

Total

3

2220

1800

6660

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

2250

1800

2250

1800

H2O

0.112

2250

1800

252

201.6

10x T4 Buffer

0.1

2250

1800

225

180

5M NaCl

0.01

2250

1800

22.5

18

1 mg/ml BSA

0.05

2250

1800

112.5

90

T4 DNA ligase

0.1675

2250

1800

376

301.

875

5

Total

1.4

2250

1800

3238

2591.

875

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_PLATE1

1

2017_ALFALFA_

PLATE2

2

2017_ALFALFA_PLATE3

3

2017_ALFALFA_PLATE4

4

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.

...

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

1

 2017_ALFALFA_PLATE1

 2017_ALFALFA_PLATE2

 2017_ALFALFA_PLATE3

 2017_ALFALFA_PLATE4

2

3

4

5

6a (cycler)

 2017_ALFALFA_PLATE10

 2017_ALFALFA_PLATE11

6b (incubator)

 2017_ALFALFA_PLATE10

 2017_ALFALFA_PLATE11

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

1100

900

10472

8568

5x iProof buffer

4

1100

900

4400

3600

10 mM dNTPs

0.4

1100

900

440

360

50 mM MgCl2

0.4

1100

900

440

360

5 uM Illumina Primers

1.33

1100

900

1463

1197

iProof TAQ

0.2

1100

900

220

180

DMSO

0.15

1100

900

165

135

total

16

1100

900

17600

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

1100

900

467

382.5

10 mM dNTPs

0.4

1100

900

440

360

Primers

1.33

1100

900

1463

1197

Total

2.155

1100

900

2370

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.