Jul 13, 2024

Public workspaceGT-seq protocol EFGL

DOI
dx.doi.org/10.17504/protocols.io.j8nlko1e6v5r/v1
  • 1Eagle Fish Genetics Lab
EagleFish GeneticsLab
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DOI: dx.doi.org/10.17504/protocols.io.j8nlko1e6v5r/v1
Protocol Citation: EagleFish GeneticsLab 2024. GT-seq protocol EFGL. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlko1e6v5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 29, 2023
Last Modified: July 13, 2024
Protocol Integer ID: 92797
Keywords: PCR, barcoding, SequalPrep, Normalization, Ampure, Qubit, NextSeq 2000, Eagle Fish Genetics Lab,
Abstract
The purpose of this protocol is to prepare extracted samples of DNA to be read on a NextSeq 2000. This will be achieved through SNP-PCR, barcoding, SequalPrep Normalization, Ampure bead size selection, and Qubit quantification. This will result in a single pooled vial of DNA ready for the NextSeq 2000 EFGL Loading Protocol.

Materials
SNP-PCR
  • Print out Library Prep Sheet from project checklist excel file
  • Qiagen Master mix (aliquots in box in refrigerator)
  • Panel specific GT-seq Primers (aliquots in box in refrigerator)
  • Low profile non-skirted PCR tray
  • 0.1mL combi-tip
  • 1000ul tips for larger master mixes
  • DNA tray
  • p10 multi-channel pipette and box of tips
  • Heat seal
  • Non-Lo Bind 1.5ml or 1.7ml tube
  • Gloves

Barcoding
  • SNP-PCR tray
  • 25ml reservoir
  • multi-channel pipette and box of tips
  • lab grade H2O
  • 3 heat seals
  • i7 tube(s)
  • i5 barcode plate(s)
  • HotStart master mix
  • low profile non-skirted PCR tray
  • 1.5ml non-Lo Bind tube
  • 2 boxes of 0.1-10ul pipette tips
  • 0.1 mL combi-tip
  • Gloves

SequalPrep Normalization
  • SequalPrep Normalization tray
  • SequalPrep binding buffer - Red
  • SequalPrep wash buffer – Blue
  • SequelPrep elution buffer —Yellow
  • 2 1.0 mL combi-tip
  • 5mL combi-tip
  • Multichannel pipette and one box of tips
  • 2 heat seals
  • Paper towels
  • 1.5mL Lo-Bind tube
  • Divided reservoir (25mL)
  • Gloves

Bead Size Selection
  • Ampure beads
  • Desktop cooler
  • 1 1.5ml tube
  • 3 1.5ml lo-Bind tubes
  • Magnet Stand
  • p100 pipette and tips
  • p1000 and tips
  • 80% ethanol, made fresh daily
  • Low TE
  • Gloves

BSS quantification with Qubit
  • BSS tube at room temperature
  • Qubit dsDNA high sensitivity kit (Buffer and florescent reagent)
  • Kimwipes
  • Tube/vial for Qubit working solution
  • Qubit assay tube
  • 1 1.5ml Lo-Bind tube
  • Gloves
Protocol materials
ReagentSequalPrep Normalization Plate Kit 96-wellThermo Fisher ScientificCatalog #A1051001
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
ReagentTE pH 8.0 (1X TE Solution)IDT TechnologiesCatalog #11-01-02-05
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
ReagentHot Start Taq 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0496L
ReagentQIAGEN Multiplex PCR KitQiagenCatalog #206145
Reagent Ampure XP beads Beckman CoulterCatalog #A63881
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
Reagentlow TEFisher ScientificCatalog #AAJ75793AP
SNP-PCR
SNP-PCR
19m 30s
19m 30s
Prepare SNP-PCR master mix using species specific primer panel.


Note
Equipment and supplies needed for this step:
Thermalcyclers (we have several thermalcyclers. Here's an example of one we use often)

Equipment
Mastercycler Pro
NAME
thermalcycler
TYPE
Eppendorf
BRAND
No Longer Manufactured
SKU
Download 20230412_092344.jpg

Equipment
EPPENDORF SCIENTIFIC CENTRIFUGE 5804
NAME
plate centrifuge
TYPE
Eppendorf
BRAND
02-262-8153PM; discontinued
SKU
Download 20230412_092725.jpg

Equipment
Repeater-M4
NAME
pipette
TYPE
Eppendorf
BRAND
14-287-150
SKU

Equipment
ALPS 25 V
NAME
manual heated plate sealer
TYPE
Thermo Scientific
BRAND
AB-0384/110; discontinued
SKU
Download 20230412_092748.jpg
p10 multi-channel pipette and tips
p1000 pipette and tips
1.5 mL microcentrifuge tube
benchtop centrifuge
0.1 mL Eppendorf combi-tips (Fisher Scientific; 13-683-703)
Vortex
Heat seals (Fisher Scientific; AB-0745)
Unskirted 96 well PCR trays (Fisher Scientific; AB-0700)

Reagents needed:
DNA tray(s)
ReagentQIAGEN Multiplex PCR KitQiagenCatalog #206145
Panel specific GT-seq primers


Vortex and spin down aliquots of Qiagen Multiplex Master Mix and panel specific GT-seq primers, keep them in benchtop coolers. The table has been formulated to account for pipette errors.



Combine the ingredients in a non- Lo-bind tube. Vortex and spin down tube and keep in benchtop cooler till ready to dispense.
Label unskirted PCR plate(s): SNP-PCR, date, tray#, project initials, and your initials.
Remove the DNA tray(s) from fridge. Briefly vortex and spin down Centrifigation DNA trays.
Using a 0.1mL combi-tip, dispense Amount5 µL of PCR master mix into each well.
Using a p10 multichannel pipette, add Amount2 µL of DNA into corresponding well. Discard tips after each use.
Heat seal the SNP-PCR plate(s). Vortex and briefly spin down Centrifigation SNP-PCR plate(s).
Run the SNP-PCR plate(s) on a thermalcycler using the following program. (In our lab, the program can be found using the menu options GTseq --> gs-fxtd+10c-65)

1. Temperature95 °C for Duration00:15:00
2. Temperature95 °C for Duration00:00:30
3. Temperature57 °C for Duration00:00:30
4. Temperature72 °C for Duration00:02:00
Repeat 2-4 for 5 cycles, with a 5% ramp
5. Temperature95 °C for Duration00:00:30
6. Temperature65 °C for Duration00:00:30
7. Temperature72 °C for Duration00:00:30
Repeat 5-7 for 10 cycles
8. Temperature4 °C indefinitely

19m 30s
Heat seal the DNA tray(s), and stored them back in the fridge.
Barcoding
Barcoding
21m 40s
21m 40s
Barcoding amplicons with i5s and i7s.


Note
Equipment and supplies needed for this step:
thermal cycler
manual heated plate sealer
plate seals
p10 multi-channel pipette and tips
p200 multi-channel pipette and tips
p1000 pipette and tips
Repeater-M4 pipette
0.1mL combi-tip (Fisher Scientific; 13-683-700)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
1.5mL microcentrifuge tubes
50mL reservoir
vortex
Plate centrifuge

Reagents needed:
SNP-PCR plate(s)
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
ReagentHot Start Taq 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0496L
aliquoted i5 tray from IDT
diluted unique i7s


After SNP-PCR thermalcycler program has completed, remove SNP-PCR plate(s). Briefly vortex and spin down Centrifigation .
Pour lab-grade water into a 50mL reservoir. Using a p200 multichannel pipette, dispense 133ul of lab grade water into each well.
Note
To prevent contamination, discard tips after each use if you don't feel comfortable pipetting over the wells.

Heat seal diluted SNP-PCR tray(s). Vortex well, and centrifuge Centrifigation3000 rpm, 00:00:30

30s
Make barcoding master mix.

IMPORTANT! Each DNA tray needs to have an unique i7, if they will be loaded on the same library.

Check that i7s have been diluted to working stock of 11uM, and proceed with preparing barcoding mix. If not, add Amount961 µL of lab-grade water to Amount119 µL of 100uM i7 to make working stock.

Vortex Hotstart MM and i7(s), spin down and store in benchtop cooler.



Combine ingredients in a 1.5mL non- Lo-bind tube(s). Vortex, spin down and store in benchtop cooler.
Label unskirted PCR plate: BARCODE, Date, Tray#, project initials and your initials.
Remove the pre-aliquot i5 trays. Briefly vortex, and spin down Centrifigation .

Using a 0.1mL combi-tip, dispense Amount6 µL of barcoding mix into each well of the Barcode plate(s).

Using a p10 multi-channel pipette, add Amount2 µL of i5 barcode into each corresponding well of the Barcode plate(s). Discard tips after each use.
Once the i5s have been added, add Amount3 µL of the diluted SNP-PCR into each corresponding well of the Barcode plate(s). Discard tips after each use.
Heat seal the Barcode plate(s). Gently vortex, and spin down Centrifigation .
Run the Barcode plate(s) on a thermalcycler using the following program. (In our lab, the program can be found using the menu options GTseq --> RAD-amp)

1. Temperature95 °C for Duration00:15:00
2. Temperature95 °C for Duration00:00:10
3. Temperature65 °C for Duration00:00:30
4. Temperature72 °C for Duration00:00:30
Repeat 2-4 for 10 cycles
5. Temperature97 °C for Duration00:05:00
6. Temperature4 °C indefinitely

21m 10s
Heat seal the i5 tray, and diluted SNP-PCR plate(s). Store them in fridge.
Sequalprep Normalization
Sequalprep Normalization
1h 5m
1h 5m
Normalize the barcoded samples with SequalPrep kit.


Note
Equipment and supplies needed for this step:
Applied Biosystems SequalPrep Normalization plates
manual heated plate sealer
plate seals
p10 multi-channel pipette and tips
Repeater-M4 pipette
1mL combi-tip
5mL combi-tip
1.5mL Lo-bind microcentrifuge tube (Fisher Scientific; 13-698-791)
25mL reservoir with divider
vortex
Plate centrifuge

Reagents needed:
Barcode plate(s)
ReagentSequalPrep Normalization Plate Kit 96-wellThermo Fisher ScientificCatalog #A1051001


After Barcode thermalcycler program has completed, remove Barcode plate(s). Briefly vortex and spin down Centrifigation .
Label a new SequalPrep Normalization plate: Date, Tray#, project initials and your initials.
Using a 1mL combi-tip, dispense Amount10 µL of Binding Buffer into each well of the SequalPrep plate(s). If you have a partial tray, only dispense binding buffer that contains barcoded samples.
Using a p10 multi-channel pipette, transfer all Amount11 µL of barcoded samples into corresponding well of the SequalPrep plate(s). Discard tips after each use.
Heat seal SequalPrep plate(s). Vortex, and briefly spin down Centrifigation .

Incubate SequalPrep plate(s) at room temperature for Duration01:00:00 . (OK to leave plate(s) at room temperature overnight if necessary)
1h
After incubation, empty out all contents from the SequalPrep plate(s) into sink.
Tap plate(s) on paper towels to remove any residual binding buffer/barcoded samples.
Using a 5mL combi-tip, dispense 50ul of Wash Buffer into each well of the SequalPrep plate(s). Avoid touching the side of the wells, change tip if you think there might be contamination.
Empty out all wash buffer from SequalPrep plate(s) into sink.
Tap plate(s) on paper towels to remove any residual wash buffer in wells.
Using a 1mL combi-tip, dispense Amount20 µL of Elution Buffer into each well of the SequalPrep plate(s). Avoid touching the side of the wells, change tip if you think there might be contamination.

Heat seal SequalPrep plate(s). Vortex, and spin down Centrifigation .

Incubate SequalPrep plate(s) at room temperature for Duration00:05:00 . (OK to store plate(s) in fridge overnight before pooling if necessary)
5m
Using a p10 multi-channel pipette, pool Amount10 µL of each sample into a 25mL reservoir. OK to reuse tips.

Transfer all pooled samples from reservoir into a 1.5mL Lo-bind tube. Label tube(s): NP# (tray#), project initials. Store in fridge.
Heat seal SequalPrep plate(s), and store in fridge.
Beads Size Selection
Beads Size Selection
32m 30s
32m 30s
Invitrogen‱ DynaMag‱- Spin Magnet
Note
Equipment and supplies needed for this step:
p10 pipette and tips
p200 pipette and tips
p1000 pipette and tips
1.5mL microcentrifuge tube
1.5mL Lo-bind microcentrifuge tube (Fisher Scientific; 13-698-791)
vortex
Invitrogen DynaMag Spin Magnet (taped in place so it does not spin)

Reagents needed:
NP tube(s)
Reagent Ampure XP beads Beckman CoulterCatalog #A63881
100% ethanol (laboratory grade, non-denatured
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
Reagentlow TEFisher ScientificCatalog #AAJ75793AP


Vortex Ampure XP beads well, and briefly spin down to avoid beads getting stuck on lid. Keep in benchtop cooler.


Note
Ampure XP beads have been aliquoted into 2 mL tubes to prevent contamination of the stock. In general, whenever they are used in the protocol, they should be vortexed thoroughly to ensure mixing

Vortex NP tube(s), and spin down. Keep in benchtop cooler.
Label 1.5mL Lo-bind tube(s) with the corresponding NP tube number, if performing multiple bead size selection simultaneously.
Add Amount25 µL of Ampure XP beads into each labeled 1.5mL Lo-Bind tube.

Add Amount50 µL of NP tube into the corresponding labeled 1.5mL Lo-bind tube containing Ampure XP beads.

Gently vortex, and quick spin down. Incubate tube(s) at room temperature for Duration00:05:00 .

Meanwhile, store the NP tube(s) back into fridge.

5m
Place tube(s) on magnetic stand for Duration00:05:00 .

5m
Carefully transfer supernatant (~Amount75 µL ) into a new 1.5mL Lo-bind tube. Try to avoid disturbing the bead pellet, and be sure to label the new tube(s).

To the supernatant, add Amount35 µL of Ampure XP beads. Repeat step if having multiple tube(s).

Gently vortex, and quick spin down. Incubate tube(s) at room temperature for Duration00:05:00 .

In the meantime, prep the 80% ethanol wash solution. Each tube will need a total of Amount400 µL 80% ethanol wash solution.
Add Amount400 µL of 100% ethanol with Amount100 µL of lab-grade water, multiply volume according to the total number of tubes you'll be doing. Vortex, spin down and set aside on benchtop.

5m
Place tube(s) on magnetic stand for Duration00:05:00 .

If time allows, store Ampure XP beads back in fridge.
5m
Transfer and discard all supernatant (~Amount110 µL ) without disturbing the bead pellet.

While on magnetic stand, gently add Amount200 µL of 80% ethanol wash solution to each tube(s).

Incubate for Duration00:00:30 , and discard supernatant. Repeat wash step one more time.

30s
While on magnetic stand, air dry beads for Duration00:05:00 . Avoid overdrying beads.

5m
Remove tube(s) from magnetic stand, and add Amount17 µL low TE. Resuspend beads by pipetting or gently vortex.
Incubate tube(s) at room temperature for Duration00:02:00 .
2m
Place tube(s) on magnetic stand for Duration00:05:00 .
5m
Carefully transfer all Amount17 µL supernatant into a new 1.5mL Lo-bind tube(s), labeled BSS#.

This is now your final, undiluted GT-seq library(s).
Quantify library
Quantify library
3m
3m
Qubit GT-seq library


Note
Equipment and supplies needed for this step:

Equipment
Qubit Fluorometer
NAME
Fluorometer to quantify DNA
TYPE
Invitrogen
BRAND
Q33226
SKU
Download 20230412_092455.jpg

Qubit assay tubes (Fisher Scientific, Q32856)
2.0mL microcentrifuge tube
1.5mL Lo-bind microcentrifuge tube (Fisher Scientific; 13-698-791)
p10 pipette and tips
p200 pipette and tips
vortex
benchtop centrifuge

Reagents needed:
BSS tube(s)
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
ReagentTE pH 8.0 (1X TE Solution)IDT TechnologiesCatalog #11-01-02-05 (not sure if the right one)


If BSS tube(s) were stored in fridge, allow tube(s) to come to room temperature prior to qubit.
Make 1:200 diluted dye mix in a 2.0mL microcentrifuge tube.



Label each qubit assay tube(s). Only label on the lid, never the side of the tube(s).
Pipette Amount198 µL of diluted dye mix into each qubit assay tube(s). Add Amount2 µL of corresponding BSS to labeled qubit assay tube(s).

Vortex thoroughly, quick spin.
Incubate tube(s) at room temperature for Duration00:03:00 .

3m
Put qubit assay tube in Qubit Fluorometer and take 2 readings per sample and enter them into Library Prep sheet in PROJECT CHECKLIST_v2.4 spreadsheet. The spreadsheet has a column that will take the average, and make the calculations in the Tray-Quant tab to dilute each library down to 1nM.
In a new 1.5mL Lo-bind tube, transfer Amount10 µL of BSS and dilute it with the calculated amount with 1X TE. Label tube as D#, include your initials and project initials.

Store diluted BSS tube(s) in GT-seq run box in fridge. Discard the undiluted BSS tube(s). Print out the Library Prep sheet when all tube(s) are ready for the run.
Protocol references
Steele, C. A., Hess, M., Narum, S., & Campbell, M. (2019). Parentage‐based tagging: Reviewing the implementation of a new tool for an old problem. Fisheries, 44(9), 412–422. https://doi.org/10.1002/fsh.10260