Wagner Trout - First 12 plates (Trout1)

As of 12-14-2021:

3700 fish DNA samples expected for Restriction Fragment Sequencing library prep. 2-3 plates will be pooled for PCR and sequencing. The first 13 plates arrived 12-13-21.

WY_YCT_2021_Plate11 on submission form, but not submitted.

WY_YCT_2021_Plate12 submitted, not on form.

 

Setup Notes

~40 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

  • Only 2-3 plates will be included in each pool

  • Pooling of samples for PCR will either only be done in groups of three or handled by half plates in sets of 4

  •  Each library should be sequenced on a 1x100bp run on a NovaSeq SP flowcell – doing this at the sequencing facility you usually use in Denver should work just fine for us. I believe you’ve previously mentioned that each run with these specifications costs approximately $3,100.

  • Please size select each library for the 300-350bp fragment size window via Pippin Prep

  • Multiplex ~3 plates-worth of individuals per library – the following arrangements of plates within libraries partially accounts for differences in sample age, extraction method, and hopefully concentration.

    • MT_YCT_RangewideRADs_plate2, WY_YCT_2021_plate7, WY_YCT_2021_plate12, and WRosenthal_plate8_Dec21 (if possible) should be together

    • WRosenthal_plate1_Dec21, WRosenthal_plate2_Dec2021, and WRosenthal_plate3_Dec21 should be together

    • WRosenthal_plate4_Dec21, WRosenthal_plate5_Dec2021, and WRosenthal_plate6_Dec21 should be together

    • WRosenthal_plate7_Dec21, GY21_YCT_tray2, and GY21_YCT_tray5 should be together

Client Plate Name

GTL Plate Name

Pool

Client Plate Name

GTL Plate Name

Pool

MT_YCT_RangewideRADs_plate2

Trout1

1

WY_YCT_2021_plate7

Trout2

1

WY_YCT_2021_plate12

Trout3

1

WRosenthal_plate8_Dec21

Trout4

1

WRosenthal_plate1_Dec21

Trout5

2

WRosenthal_plate2_Dec2021

Trout6

2

WRosenthal_plate3_Dec21

Trout7

2

WRosenthal_plate4_Dec21

Trout8

3

WRosenthal_plate5_Dec2021

Trout9

3

WRosenthal_plate6_Dec21

Trout10

3

WRosenthal_plate7_Dec21

Trout11

4

GY21_YCT_tray2

Trout12

4

GY21_YCT_tray5

Trout13

4

Check In Samples Against List from Will Rosenthal

Load Submission Data into MISO

Quantify and normalize samples:

  • Normalize plate reader with Buffer AE for all plates

  • Quantify Trout5 - Trout13

  • Check for samples above 150 ng/ul

  • Transfer 20 ul from all wells of a plate needing normalization to new VWR plate

  • Add 20 ul TE to all wells above 150 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 11 plates using the 8 channel

Reagent

ul/rxn

rxns

ul needed (1.5x)

10x T4 Buffer

1.15

1160

2001

5M NaCl

0.12

1160

209

1 mg/ml BSA

0.6

1160

1044

H2O

0.73

1160

1270

MseI (enzyme)

0.12

1160

209

EcoR1 (enzyme)

0.28

1160

487

Total

3

1160

5220

Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.

Reagent

ul/rxn

rxns

ul needed (1.6x)

Reagent

ul/rxn

rxns

ul needed (1.6x)

MseI oligo

1

1160

1857

H2O

0.112

1160

208

10x T4 Buffer

0.1

1160

186

5M NaCl

0.01

1160

18.6

1 mg/ml BSA

0.05

1160

93

T4 DNA ligase

0.1675

1160

311

Total

1.4

1160

2673.6

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.

Template

EcoR1 MID plate

Pool

Template

EcoR1 MID plate

Pool

Trout1

1

1

Trout2

2

1

Trout3

3

1

Trout4

4

1

Trout5

5

2

Trout6

6

2

Trout7

7

2

Trout8

8

3

Trout9

1

3

Trout10

2

3

Trout11

3

4

Trout12

4

4

Trout13

5

4

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.

Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Create working pools. For Pool A combine ligated plates matching well to well. For pool B combine ligated plates with even plates flipped upside down (H12 in A1). C1 and C2 will be recombining Plate 5 column wise. Combine columns 1-3, 4-6, 7-9 and 10-12 into columns 1-3 of a new plate. Then, to columns 7-9 of the same plate, add columns 1-3 and 7-9. Flip Plate 5 around and then pipette 6-4 and 12-10 into columns 7-9 of the pool plate. Mix together 20 ul from each well of each plate for pooled plate using the Benchsmart. After pooling all samples, vortex and spin down plates then use 4 ul from the pooled plates for PCR templates.

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

1A

Trout1

Trout2

Trout3

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

1B

Trout1

Trout2

(FLIPPED)

Trout3

 

Pool

Wells A1-D1

Wells E1-G1

Pool

Wells A1-D1

Wells E1-G1

1C

Trout4 Wells A1-D1

Trout4 E1-G1

 

Pool

Wells A1-C1

Wells D1-G1

Pool

Wells A1-C1

Wells D1-G1

1D

Trout4 Wells A1-C1

Trout4 D1-G1

 

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

2A

Trout5

Trout6

Trout7

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

2B

Trout5

Trout6

(FLIPPED)

Trout7

 

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

3A

Trout8

Trout9

Trout10

 

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

3B

Trout8

Trout9

(FLIPPED)

Trout10

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

4A

Trout11

Trout12

Trout13

Pool

Plate1

Plate2

Plate3

Pool

Plate1

Plate2

Plate3

4B

Trout11

Trout12

(FLIPPED)

Trout13

PCR1:

Reagent

ul/rxn

rxns

ul needed (x1.4)

Reagent

ul/rxn

rxns

ul needed (x1.4)

H2O

9.52

770

10,272

5x iProof buffer

4

770

4316

10 mM dNTPs

0.4

770

432

50 mM MgCl2

0.4

770

432

5 uM Illumina Primers

1.33

770

1435

iProof TAQ

0.2

770

216

DMSO

0.15

770

162

total

16

770

16,833

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel

Add 4uL of template from pooled plates with Benchsmart

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

Temp C

Cycles

Time

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 2.155 ul of the MM directly to the previous PCR

Reagent

ul/rxn

rxns

ul needed (x 1.6)

5x Iproof buffer

0.425

770

524

10 mM dNTPs

0.4

770

493

Primers

1.33

770

1639

Total

2.155

770

2656

Then continue with the last four unlisted steps for GBS1 from above:

Temp C

Cycles

Time

Temp C

Cycles

Time

98

1X

3:00

60

1X

2:00

72

1X

10:00

4

1X

0:00

Pippin Prep Size Select (350-450 bp select):

 

Run Final Product on qPCR for check

Result from qPCR check:

 

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