Wagner Trout 2 (2Trout)

Setup Notes

~32 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

Pool ~4*96 individuals per library
Cleanup each full pool via ultra purification
Size select for 300-366bp fragments size window via BluePippin or Pippin Prep
Sequence each library on a NextSeq P3 flowcell
5% PhiX spike

Client Plate Name	GTL Plate Name	Low Volume Wells	Empty Wells	EcoR1	Samples

Client Plate Name	GTL Plate Name	Low Volume Wells	Empty Wells	EcoR1	Samples
WRosenthal_Trout_plate9	WRT9			1	96
WRosenthal_Trout_plate10	WRT10			2	96
WRosenthal_Trout_plate11	WRT11			3	96
WRosenthal_Trout_plate12	WRT12			4	96
WRosenthal_Trout_plate13	WRT13		B8	5	95
WRosenthal_Trout_plate14	WRT14			6	96
WRosenthal_Trout_plate15	WRT15			7	96
WRosenthal_Trout_plate16	WRT16			8	96
WRosenthal_Trout_plate17	WRT17		D5, F5	1	94
WRosenthal_Trout_plate18	WRT18			2	96
WRosenthal_Trout_plate19	WRT19			3	96
WRosenthal_Trout_plate20	WRT20			4	96
WRosenthal_Trout_plate21	WRT21		G11, H11	5	94
WRosenthal_Trout_plate22	WRT22			6	96
WRosenthal_Trout_plate24	WRT24		H6, G12, H12	7	93
MT_YCT_RangewideRADs_plate01	WR_MYR1			8	96
MT_YCT_RangewideRADs_plate03	WR_MYR3			1	96
WY_YCT_2021_plate04	WR_WYCT4		E12, F12	2	94
WY_YCT_2021_plate09	WR_WYCT9			3	96
WY_YCT_2021_plate10	WR_WYCT10			4	96
WY_YCT_2021_plate11	WR_WYCT11			5	96
WY_YCT_2021_plate13	WR_WYCT13			6	96
Wrosenthal_GY21_YCT_tray1	WR_GYCT1	G1, H1, C9, F9, H9, F10, A12-D12		7	86
Wrosenthal_GY21_YCT_tray3	WR_GYCT3	H1		8	95
Wrosenthal_GY21_YCT_tray4	WR_GYCT4	A10		1	95
Wrosenthal_GY21_YCT_tray6	WR_GYCT6			2	96
Wrosenthal_PM_YCT_plate2	WR_PMCT2	H4, B8		3	94
Wrosenthal_PM_YCT_plate1	WR_PMCT1		H4, Col 5, A6-E6	4	82
WRosenthal_Trout_plate25	WRT25		C6-H6, H9, Col 10, 11, 12	5	65
MT_WCT_plate15	WR_WCT15		E8-H8, Col 9, 10, 11, 12	6	60
Wrosenthal_GY21_YCT_tray7	WR_GYCT7		H8, Col 9, Col 10, Col 11, A12-G12	7	63
WY_YCT_2021_plate14	WR_WYCT14		Col 11, 12	8	80

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 all plates
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 32 plates using the 8 channel

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	3072	5,299.2
5M NaCl	0.12	3072	553
1 mg/ml BSA	0.6	3072	2764.8
H2O	0.73	3072	2242.6
MseI (enzyme)	0.12	3072	553
EcoR1 (enzyme)	0.28	3072	1290.3
Total	3	3072	12702.9

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	3072	4915.2
H2O	0.112	3072	550.5
10x T4 Buffer	0.1	3072	491.5
5M NaCl	0.01	3072	49.2
1 mg/ml BSA	0.05	3072	245.8
T4 DNA ligase	0.1675	3072	823.3
Total	1.4	3072	7075.5

Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.

Template	EcoR1 MID plate	Pool Plate	Library

Template	EcoR1 MID plate	Pool Plate	Library
WRosenthal_Trout_plate9	1	2Trout_Pool1-4	1
WRosenthal_Trout_plate10	2	2Trout_Pool1-4	1
WRosenthal_Trout_plate11	3	2Trout_Pool5-8	1
WRosenthal_Trout_plate12	4	2Trout_Pool5-8	1
WRosenthal_Trout_plate13	5	2Trout_Pool9-12	2
WRosenthal_Trout_plate14	6	2Trout_Pool9-12	2
WRosenthal_Trout_plate15	7	2Trout_Pool13-16	2
WRosenthal_Trout_plate16	8	2Trout_Pool13-16	2
WRosenthal_Trout_plate17	1	2Trout_Pool21-24	4
WRosenthal_Trout_plate18	2	2Trout_Pool21-24	4
WRosenthal_Trout_plate19	3	2Trout_Pool25-28	4
WRosenthal_Trout_plate20	4	2Trout_Pool25-28	4
WRosenthal_Trout_plate21	5	2Trout_Pool17-20	3
WRosenthal_Trout_plate22	6	2Trout_Pool17-20	3
WRosenthal_Trout_plate24	7	2Trout_Pool53-56	3
WRosenthal_Trout_plate25	8	2Trout_Pool53-56	3
MT_YCT_RangewideRADs_plate01	1	2Trout_Pool29-32	5
MT_YCT_RangewideRADs_plate03	2	2Trout_Pool29-32	5
WY_YCT_2021_plate04	3	2Trout_Pool33-36	5
WY_YCT_2021_plate09	4	2Trout_Pool33-36	5
WY_YCT_2021_plate10	5	2Trout_Pool37-40	6
WY_YCT_2021_plate11	6	2Trout_Pool37-40	6
WY_YCT_2021_plate13	7	2Trout_Pool49-52	6
WY_YCT_2021_plate14	8	2Trout_Pool49-52	6
Wrosenthal_GY21_YCT_tray1	4	2Trout_Pool45-48	8
Wrosenthal_GY21_YCT_tray3	5	2Trout_Pool45-48	8
Wrosenthal_GY21_YCT_tray4	6	2Trout_Pool61-64	8
Wrosenthal_GY21_YCT_tray6	7	2Trout_Pool57-60	7
Wrosenthal_PM_YCT_plate2	3	2Trout_Pool41-44	7
Wrosenthal_PM_YCT_plate1	2	2Trout_Pool41-44	7
MT_WCT_plate15	1	2Trout_Pool61-64	8
Wrosenthal_GY21_YCT_tray7	8	2Trout_Pool57-60	7

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 2 Working Pool versions for all samples within full plate or close to full plates by combining column 1, 4, 7, and 9 into column 1 or 7 of the pool plate; 2, 5, 8, and 10 into column 2 or 8; and column 3, 6, 9, and 12 into column 3 or 9. Pool plate column 4 or 10 receive columns 1, 2, 3, and 4. Pool plate column 5 or 11 receive columns 5, 6, 7, and 8. Pool plate column 6 or 12 receive columns 9, 10, 11, and 12.

PCR1:

Reagent	ul/rxn	rxns	ul needed (x1.4)

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	576	7712	5483.52	1904
5x iProof buffer	4	576	3240	2304	800
10 mM dNTPs	0.4	576	324	230.4	80
50 mM MgCl2	0.4	576	324	230.4	80
5 uM Illumina Primers	1.33	576	1077	766.08	266
iProof TAQ	0.2	576	162	115.2	40
DMSO	0.15	576	121	86.4	30
total	16	576	12960	9216	3200

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	1152	784	163.2
10 mM dNTPs	0.4	1152	738	153.6
Primers	1.33	1152	2452	511
Total	2.155	1152	3973.82	828

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

Pool Using PCR Plates with Assist Plus

Organize plates by Library # above, vortex, and quick spin

Use 4GbsPoolx8

Use single channel to combine 48 ul from each well of a column into a separate labeled tube.

Pippin Prep Size Select (300-366 bp select):

Run Final Product on qPCR for check and quant

Result from qPCR check:

load =800 pM

dilute 1 nM PhiX to 500 pM

Library	qPCR Result (nM)	RSB ul for 2nM	Library ul for 2 nM	RSB ul for load	Library ul for Load	PhiX ul for load	Loading Concentration Used	% Occupied	Samples	Yield Gbp

Library	qPCR Result (nM)	RSB ul for 2nM	Library ul for 2 nM	RSB ul for load	Library ul for Load	PhiX ul for load	Loading Concentration Used	% Occupied	Samples	Yield Gbp
1	6.5	34.6	15.4	10.4	8	1.6			384
2	4.7	28.7	21.3	10.4	8	1.6			383
3	4.9	29.6	20.4	10.4	8	1.6			348
4	5.3	31.1	18.9	10.4	8	1.6			382
5	6	33.3	16.7	10.4	8	1.6			374
6	5.5	31.8	18.2	10.4	8	1.6			365
7	3.9	24.4	25.6	11.4	7	1.6	700pM		336
8	7.6	36.8	13.2	10.4	8	1.6	800 pM	98.97	297	126.52
1SMIN							900	99.23		134.84

Genome Technologies Laboratory