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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)

Original SOP

Normalize templates

Use plate reader to quantify templates

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

...

Seal, vortex, and spin tubes. You will need 2 tubes for this prep.

MseI oligo Annealing

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

...

Heat to 95C for 5 minutes and allow to slowly cool to RT.

EcoRI plate 4 oligo Annealing

SpeedVac plates Stock Plate 7 and Stock Plate 8 to dry oligos

Resuspend plate 4a and 4b to 25 uM. We will have to calculate based off initial expected volume and concentrationwith 20 ul H2O and 60 ul std TE to avoid over saturating with Tris and EDTA to create 25 uM stocks.

Combine 4 ul from each plate in a well to well fashion with 92 ul of TE to create a 1 uM working plate.

Label it “GBS Working Stock Plate 4” Seal, Vortex, and Spin down resulting plate.

Heat to 95C for 5 minutes and allow to slowly cool to RT

Restriction Digestion

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 22 16 plates using the Benchsmart

Reagent

ul/rxn

rxns

ul needed

10x T4 Buffer

1.15

2400

1800

2760

2070

5M NaCl

0.12

2400

1800

288

216

1 mg/ml BSA

0.6

2400

1800

1440

1080

H2O

0.73

2400

1800

1752

1314

MseI (enzyme)

0.12

2400

1800

288

216

EcoR1 (enzyme)

0.28

2400

1800

672

504

Total

3

2400

1800

7200

5400

Use an 8 channel to divvy up 71 ul across a plate and then the Benchsmart to add 3 ul to 22 plates.

Add 6 ul template to each plate with 8 channel. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 62C for 1 hour.

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with Benchsmart.

Reagent

ul/rxn

rxns

ul needed

MseI oligo

1

2400

1800

2400

1800

H2O

0.112

2400

1800

268

201.

8

6

10x T4 Buffer

0.1

2400

1800

240

180

5M NaCl

0.01

2400

1800

24

18

1 mg/ml BSA

0.05

2400

1800

120

90

T4 DNA ligase

0.1675

2400

1800

402

301.5

Total

1.4

2400

1800

3454

2591.

8

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_

PLATE12017_ALFALFA_PLATE2

PLATE5

5

2017_ALFALFA_

PLATE32017_ALFALFA_PLATE4

PLATE6

6

2017_ALFALFA_

PLATE52017_ALFALFA_PLATE6

PLATE7

7

2017_ALFALFA_

PLATE72017_ALFALFA_PLATE82017_ALFALFA_PLATE11

PLATE8

8

2017_ALFALFA_PLATE9

2017_ALFALFA_PLATE10

1

Label reaction plates with MID plate used.

...

Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

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

6

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

700

900

6664

8568

5x iProof buffer

4

700

900

2800

3600

10 mM dNTPs

0.4

700

900

280

360

50 mM MgCl2

0.4

700

900

280

360

5 uM Illumina Primers

1.33

700

900

931

1197

iProof TAQ

0.2

700

900

140

180

DMSO

0.15

700

900

105

135

total

16

700

900

10400

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

700

900

297

382.5

10 mM dNTPs

0.4

700

900

280

360

Primers

1.33

700

900

931

1197

Total

2.155

700

900

1508

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

Blue Pippin?

Gel Extraction?We utilized the Pippin Prep machine on loan from the Ernest Lab, selecting for the range 300-450bp.