Wagner Fish RFS Project 1 (Trout1)

As of 08-02-2021:

1000 fish DNA samples expected for Restriction Fragment Sequencing library prep. They will be split into 2 relatively even pools to avoid read count disparity and allow resolution of each sample.

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 Pool will be run by itself on an SP 1x100 NovaSeq

Check In Samples Against List from Will Rosenthal

Load Submission Data into MISO

Quantify DNA samples and normalize if any are higher than 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	1000	1725
5M NaCl	0.12	1000	180
1 mg/ml BSA	0.6	1000	900
H2O	0.73	1000	1095
MseI (enzyme)	0.12	1000	180
EcoR1 (enzyme)	0.28	1000	420
Total	3	1000	4500

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)
MseI oligo	1	1000	1600
H2O	0.112	1000	179.2
10x T4 Buffer	0.1	1000	160
5M NaCl	0.01	1000	16
1 mg/ml BSA	0.05	1000	80
T4 DNA ligase	0.1675	1000	268
Total	1.4	1000	2240

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

Template	EcoR1 MID plate	Pool
		1
		1
		1
		1
		1
		1
		2
		2
		2
		2
		2

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	Plate4
1A

Pool	Plate1	Plate2	Plate3	Plate4
1B

Pool	Plate5 Col 1-6	Plate5 Col 7-12	Plate6 Col 1-6	Plate6 Col 7-12
1C

Pool	Plate5 Col 1-6	Plate5 Col 12-7	Plate6 Col 1-6	Plate6 Col 12-7
1D

Pool	Plate7	Plate8	Plate9	Plate10
2A

Pool	Plate7	Plate8	Plate9	Plate10
2B

Pool	Plate11 Col 1-3	Plate11 Col 4-6	Plate11 Col 7-9	Plate11 Col 10-12
2C

Pool	Plate11 Col 1-3	Plate11 Col 6-4	Plate11 Col 7-9	Plate11 Col 12-10
2D

PCR1:

Make MM1 in a 15 ml tube:

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	500	6664
5x iProof buffer	4	500	2800
10 mM dNTPs	0.4	500	280
50 mM MgCl2	0.4	500	280
5 uM Illumina Primers	1.33	500	931
iProof TAQ	0.2	500	140
DMSO	0.15	500	105
total	16	500	11,200

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
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 (x 1.6)
5x Iproof buffer	0.425	240	340
10 mM dNTPs	0.4	240	320
Primers	1.33	240	1064
Total	2.155	240	1724

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

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

Run Final Product on qPCR for check

Result from qPCR check: