hyRADccg wetlab protocol

This markdown document can be printed for use at the bench with no formatting worries (such as lines spanning pages).
The reagent-calc.md doc that is also in this repository details how we calculated the amounts of probes, blocking oligos, binding beads, and input library used in this protocol.
Protocols presented here were based on those originally described by Peterson et al. 2012 and Suchan et al. 2016.

1. ddRAD RAD library & probe construction
1.1 Double Digest
1.2 Double Digest Zymo Clean-Up
1.3 Ligation
1.4 Ligation Bead Clean-Up
1.5 Size Selection
1.6 RT PCR
1.7 PCR Clean-up Using Gel Excision
1.8 QC
1.9 Deadapterization
1.10 Deadapterization Bead Clean-Up
1.11 Probe Biotinylation
1.12 Biotinylation Zymo Clean-Up
1.13 QC
2. Whole-genome Library Preps (for captures)
2.1 Covaris shearing (for modern samples only)
2.2 KAPA End Repair and A-tailing
2.3 KAPA Ligation
2.4 KAPA Ligation Bead Clean-up
2.5 RT-PCR
2.6 PCR Bead Clean-up
2.7 QC
3. Hybridization
3.1 Prepare Hybridization Mix & Blockers Mix
3.2 Incubation
3.3 Prepare Wash & Binding Buffers
3.4 Dynabead Preparation
3.5 Bead Binding & Washing
3.6 RT-PCR
3.7 PCR Bead Clean-up
3.8 QC & Final Pool

1. ddRAD RAD library & probe construction

Use the below protocol or your favorite other method for construction of a ddRAD library.

1.1 Double Digest

Quantify extracted DNA using Qubit dsDNA High Sensitivity kit
Prepare a 40 µl aliquot of DNA at concentration 12.5 ng/µl
To this aliquot, add the following reagents:

Reagent	x1
H20	2 µL
10X NEB Cutsmart buffer	5 µL
MluCI (10000 U/ml)	2 µL
SphI (20000 U/ml)	1 µL
TOTAL	50 µL

Pipette mix 5X
Using a thermal cycler, incubate the reaction at 37°C for 3 hours

1.2 Double Digest Zymo Clean-Up

using DNA Clean & Concentrator™-5 columns (Zymo Research)
- https://www.zymoresearch.com/dna/dna-clean-up/pcr-dna-clean-up-concentration/dna-clean-concentrator-5

Zymo Protocol:

Transfer digested sample to 1.5 ml non-stick tube

add 5X DNA Binding Buffer (50 µL * 5 = 250 µL DNA Binding Buffer)
pipette mix 5X and transfer sample to Zymo Clean & Concentrator column
spin at 4000 x g for 3 min
spin at 10,000 x g for 30 sec
discard flow-through
add 200 µL Wash Buffer
spin at 10,000 x g for 1 min
add 200 µL Wash Buffer
spin at 10,000 x g for 1 min 30 sec
transfer column to new tube
add 35 µL 10 mM Tris-HCl, pH 8 (pre-heated to 65°C)
incubate at room temp 3 min
spin at 10,000 x g for 1 min
- Quantify cleaned products using Qubit dsDNA High Sensitivity kit

1.3 Ligation

In a PCR tube, prepare a 30 µl aliquot of digested DNA at concentration 4 ng/µl using sterile H20

To this aliquot, add the following reagents:

Reagent	x1
10X NEB T4 DNA Ligase buffer	4 µL
10 uM PI adapter	2 µL
10 uM P2 adapter	2 µL
T4 Ligase	2 µL
TOTAL	40 µL

Pipette mix 5X
Place rxn in thermal cycler and run the following program:

Step	Temperature (°C)	Time
1	37	30 min
2	65	10 min
3	65	1.5 min, decrease -2°C every cycle for 23 cycles
4	20	hold

1.4 Ligation Bead Clean-Up

Perform standard 1.6X AmPure bead clean-up using the following protocol:

bring AmPure beads to room temperature

mix beads well
add 64 µL of beads to ligation rxn
pipette mix 10X
incubate at room temp for 10 min
place rxn on magnetic plate for 2 min
aspirate cleared solution and discard
dispense 200 µL of 80% ethanol to sample
wait 10 sec
aspirate out the ethanol and discard
repeat ethanol wash for a total of two washes
ensure all ethanol is removed from sample
place sample (on magnetic rack) in the fume hood to dry for 8 min
check that all ethanol has evaporated from the sample
remove sample from magnetic plate
resuspend beads in 30 µl of 10 mM Tris-HCl, pH 8
pipette mix 10X
incubate at room temp for 5 min
place sample on magnetic plate for 2 min
aspirate eluted DNA and transfer to a 0.5 ml non-stick tube

1.5 Size Selection

Perform a tight 325 bp target size selection using Blue Pippin 2% gel with marker V1

Proceed immediately to RT-PCR

1.6 RT PCR

using HiFi Real-Time PCR Library Amplification Kit (Kapa Biosystems)
- https://www.kapabiosystems.com/product-applications/products/next-generation-sequencing-2/library-amplification/
pipette measure exactly how much product was collected from the Pippin
divide this number in half and set up two PCR reactions per sample using the following recipe:

KAPA real time PCR reaction mix:

Reagent	x1
size selected Pippin product*	~20 µL
H2O	3 µL
KAPA HiFi HotStart Real-time PCR Master Mix (2X)	25 µL
25 uM PCR1 primer	1 µL
25 uM PCR2 indexed primer	1 µL
TOTAL	50 µL

RT-PCR Reaction Conditions:

Step	Temp (°C)	Time
(1)	98	45sec
(2)	98	15sec
(3)	65	30sec
(4)	72	30sec
plate read
(5)	72	10sec
(6)	Go to (2)

Run the PCR as many cycles as it takes for amplification to max out (plateau)
Once the amplification graph shows RFU plateau, wait until step (5) to pause or cancel the run at 72°C and remove samples
Clean PCR product using gel excision with the Zymoclean Gel DNA Recovery kit

1.7 PCR Clean-up Using Gel Excision

with Zymoclean DNA Recovery kit
- https://www.zymoresearch.com/dna/dna-clean-up/gel-dna-recovery/zymoclean-gel-dna-recovery-kit

Excison Protocol:

run PCR product out on a 1% low melt agarose gel along with a 100 bp ladder

excise the PCR band using a sterile razor and place the gel slice in a 1.5 ml non-stick tube
add 3 volumes of Buffer ADB to the tube
incubate the tube at 50°C for 10 min to dissolve the gel slice
transfer the melted gel sample to a Zymoclean DNA Recovery column
spin at 10,000 x g for 30 sec
discard the flow-through
add 200 µL Wash Buffer
spin at 10,000 x g for 30 sec
add 200 µL Wash Buffer
spin at 13,000 x g for 1 min 30 sec
transfer column to new tube
add 30 µL 10 mM Tris-HCl, pH 8 (pre-heated to 65°C)
incubate at room temp 3 min
spin at 10,000 x g for 1 min

1.8 QC

Quantify products using Qubit dsDNA High Sensitivity kit
Make a 1:20 dilution of RAD library in sterile H20
Run 1 µL of this diluted product on Bioanalyzer using the High Sensitivity DNA kit
Set aside at least 5 µL of RAD library and store at -20°C (excess product that is set aside at this step may be used to generate more probes via PCR if needed in the future)

1.9 Deadapterization

Calculate out how many ngs of product you have
Prepare as many deadapterization rxns that your product will allow for using the below recipe:

Reagent	x1
H20	var µL
10X NEB Cutsmart buffer	5 µL
MluCI (10000 U/ml)	3.30 µL
SphI (20000 U/ml)	1.70 µL
DNA	up to 1 µg
TOTAL	50 µL

Pipette mix 5X
Quick spin
Place in thermal cycler and run the following program:

Step	Temperature (°C)	Time
1	37	3 hours
2	65	20 min
3	8	hold

1.10 Deadapterization Bead Clean-Up

Perform standard 1.5X AmPure bead clean-up using the following protocol:

bring AmPure beads to room temperature

mix beads well
add 75 µL of beads to ligation rxn
pipette mix 10X
incubate at room temp for 10 min
place rxn on magnetic plate for 2 min
aspirate cleared solution and discard
dispense 200 µL of 80% ethanol to sample
wait 10 sec
aspirate out the ethanol and discard
repeat ethanol wash for a total of two washes
ensure all ethanol is removed from sample
place sample (on magnetic rack) in the fume hood to dry for 8 min
check that all ethanol has evaporated from the sample
remove sample from magnetic plate
resuspend beads in 30 µl of 10 mM Tris-HCl, pH 8
pipette mix 10X
incubate at room temp for 5 min
place sample on magnetic plate for 2 min
aspirate eluted DNA and transfer to 0.5 ml non-stick tube
Quantify products using Qubit dsDNA High Sensitivity kit
Run 1 µL of this product on Bioanalyzer using the High Sensitivity DNA kit

1.11 Probe Biotinylation

using BioNick™ DNA Labeling System (Thermo Fisher Scientific)
- https://www.thermofisher.com/order/catalog/product/18247015
Prepare as many biotinylation rxns that your product will allow for using the below recipe:

Reagent	x1
10X dNTP Mix	5 µL
DNA	up to 1 µg
H2O	var µL
10X Enzyme Mix	5 µL
TOTAL	50 µL

mix well

quick spin
ensure heated lid is 10°C over incubation temperature
incubate at 16°C for 1.5 hours
add 5 µL Stop Buffer
immediately clean reactions using Zymo Clean & Concentrator protocol below

1.12 Biotinylation Zymo Clean-Up

using DNA Clean & Concentrator™-5 columns (Zymo Research)
- https://www.zymoresearch.com/dna/dna-clean-up/pcr-dna-clean-up-concentration/dna-clean-concentrator-5

Zymo Protocol:

transfer digested sample to 1.5 ml non-stick tube
add 5X DNA Binding Buffer (50 µL * 5 = 250 µL DNA Binding Buffer)

spin at 4000 x g for 3 min
spin at 10,000 x g for 30 sec
discard flow-through
add 200 µL Wash Buffer
spin at 10,000 x g for 1 min
add 200 µL Wash Buffer
spin at 10,000 x g for 1 min 30 sec
transfer column to new tube
add 25 µL of 10 mM Tris-HCl, pH 8 (pre-heated to 65°C)
incubate at room temp 2 min.
spin at 10,000 x g for 1 min
keep cleaned product on ice
- probes can be stored at -20°C in Tris buffer for at least one year

1.13 QC

Quantify probe products using Qubit dsDNA High Sensitivity kit
Run 1 µL of this product on Bioanalyzer using the High Sensitivity DNA kit

Calculating probe amount for captures

X µL of probes at Qubit concentration (ng/µL) = Z ng probes

Add as many probes as you can given the amount of probes that you have, the number of hybridizations you want to do, and the amount of library that you have. We added ~ 100 ng of probes to each hybridization.

2. Whole-genome Library Preps (for captures)

Use the below protocol or your favorite other method that yields whole-genome libraries with indexed Illumina adapters.

using KAPA Hyper Prep kit
- https://www.kapabiosystems.com/product-applications/products/next-generation-sequencing-2/dna-library-preparation/kapa-hyper-prep-kits/

2.1 Covaris shearing (for modern samples only)

For each sample, prepare a 130 µl aliquot of DNA at concentration 7.7 ng/µl in 10 mM Tris-HCl, pH 8
Transfer sample to a Covaris microTUBE (AFA Fiber Snap-Cap)
Use the following shear settings:

Setting	value
Peak Incident Power	50
Duty Factor	20%
Cycles per Burst	200
Treatment Time	200 sec
Temperature	20°C

These settings produce sheared product in the range of 100 - 400 bps

Run 20 ul of sheared product on a standard 1.0% agarose gel along with a 100 bp ladder to confirm size of sheared product

2.2 KAPA End Repair and A-tailing

(refer to the detailed Technical Data Sheet supplied with the KAPA Hyper Prep kit)

In a standard PCR strip tube, assemble the following per sample:

Reagent	x1
Sheared DNA	50 µl
End Repair and A-tail buffer	7 µL
End Repair and A-tail enzyme mix	3 µL
TOTAL	60 µL

Pipette mix 5X
Quick spin
Place in thermal cycler and run the following program:

Step	Temp (°C)	Time
(1)	20	30 min
(2)	65	30 min
(3)	8	hold

2.3 KAPA Ligation

To each End Repaired and A-tailed reaction, add the following:

Reagent	x1
End Repaired & A-tailed product	60 µL
H20	5 µL
Ligation Buffer	30 µL
15 uM TruSeq indexed adapter*	5 µL
DNA Ligase	10 µL
TOTAL	110 µL

Pipette mix 5X
Quick spin
For modern samples, incubate the reaction at 20°C for 15 min
For ancient samples, incubate the reactions at 20°C for 4 hours

2.4 KAPA Ligation Bead Clean-up

Perform standard 1.5X AmPure bead clean-up using the following protocol:

bring AmPure beads to room temperature

transfer samples to 1.5 ml non-stick tubes
mix beads well
add 165 µL of beads to ligation rxn
pipette mix 10X
incubate at room temp for 10 min
place rxn on magnetic stand for 2 min
aspirate cleared solution and discard
dispense 200 µL of 80% ethanol to sample
wait 10 sec
aspirate out the ethanol and discard
repeat ethanol wash for a total of two washes
ensure all ethanol is removed from sample
place sample (on magnetic stand) in the fume hood to dry for 8 min
check that all ethanol has evaporated from the sample
remove sample from magnetic stand
resuspend beads in 40 µl of 10 mM Tris-HCl, pH 8
pipette mix 10X
incubate at room temp for 5 min
place sample on magnetic stand for 2 min
aspirate eluted DNA and transfer to 0.5 ml non-stick tube

2.5 RT-PCR

using HiFi Real-Time PCR Library Amplification Kit (Kapa Biosystems)
- https://www.kapabiosystems.com/product-applications/products/next-generation-sequencing-2/library-amplification/

KAPA real time PCR reaction mix

Divide library product in half to allow for two PCR rxns per sample*

Reagent	x1
KAPA Hyper DNA library product*	20 µL
KAPA HiFi HotStart Real-time PCR Master Mix (2X)	25 µL
KAPA Illumina Library Amplification Primer Mix (10X)	5 µL
TOTAL	50 µL

RT-PCR Reaction Conditions

Step	Temp (°C)	Time
(1)	98	45sec
(2)	98	15sec
(3)	60	30sec
(4)	72	30sec
plate read
(5)	72	10sec
(6)	Go to (2) 44X
(7)	72	1min

Run the PCR as many cycles as it takes for amplification to reach between 3500 - 4000 RFU. This should take only ~ 5 cycles.
Once the desired amplification level is met, wait until step (5) to pause or cancel the run at 72°C and remove samples
Combine the PCR replicates together and proceed to bead clean-up

2.6 PCR Bead Clean-up

Perform standard 1.0X AmPure bead clean-up using the following protocol:

bring AmPure beads to room temperature

mix beads well
add an equal amount of beads to PCR rxn (~100 ul)
pipette mix 10X
incubate at room temp for 10 min
place rxn on magnetic plate for 2 min
aspirate cleared solution and discard
dispense 200 µL of 80% ethanol to sample
wait 10 sec
aspirate out the ethanol and discard
repeat ethanol wash for a total of two washes
ensure all ethanol is removed from sample
place sample on magnetic rack in the fume hood to dry for 8 min
remove sample from magnetic plate
check that all ethanol has evaporated from tubes
resuspend beads in 40 µl of 10 mM Tris-HCl (pH 8)
pipette mix 10X
incubate at room temp for 5 min
place sample on magnetic plate for 2 min
aspirate eluted DNA and transfer to 0.5 ml non-stick tube

2.7 QC

Quantify products using Qubit dsDNA High Sensitivity kit
Make a 1:5 dilution of each library in sterile H20
Run 1 µl of diluted products on Bioanalyzer using the High Sensitivity DNA kit

For single sample captures:

use between 100 - 150 ng of each library for hybridization

For pooled sample captures, do the following:

Make an 18 ul working aliquot of each sample at 8.2 ng/ul in 10 mM Tris-HCl, pH 8
Pool each of the 18 ul aliquots together
Pipette mix 10X
Use a speed vac to dry down the pooled sample to ~ 7-10 ul volume
Use this pooled, concentrated sample for hybridization

3. Hybridization

Reagents needed

20X SSC
50X Denhardt’s Solution
0.5M EDTA, pH 8.0
10% Sodium Dodecyl Sulfate (SDS)
TS-p5 and TS-p7 xGen® blocking oligos (Integrated DNA Technologies)
- https://www.idtdna.com/pages/products/nextgen/target-capture/xgen-blocking-oligos
Chicken Hybloc™ DNA (Applied Genetics)
- supplied at 500 µg quantities at 1mg/ml in T.E
- http://www.appliedgenetics.com/products/hybloc-competitor-dna/
10% Sodium Dodecyl Sulfate (SDS)
H2O (molecular biology grade)
0.1X SSC, 0.1% SDS
1 M NaCl
10 mM Tris-HCl, pH 8
1 mM EDTA
10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0
Dynabeads® MyOne™ Streptavidin C1 beads (Thermo Fisher Scientific)
- https://www.thermofisher.com/order/catalog/product/65001

Other Notes

Before starting make sure bench top incubator (w/ rotator inside) is set to 55°C
Also be sure one tube incubator is set to 55°C and the other is set to 95°C
Perform captures in 1.5 ml screw-cap tubes with o-ring

3.1 Prepare Hybridization Mix & Blockers Mix

Hybridization Mix Recipe

This includes comparison with the final concentration of the reagents in the original hyRAD protocol and in the MyBaits protocol.

Reagent	x1	Final conc in 121 µL	original hyRAD protocol final concentration	MyBaits protocol final concentration
20X SSC	54 µL	8.93X	8.60X	9.47X
50X Denhardt’s Solution	21 µL	8.68X	2.87X	9.21X
0.5M EDTA, pH 8.0	3 µL	0.0124 M	0.0072 M	0.0132 M
10% Sodium Dodecyl Sulfate (SDS)	3 µL	0.248%	0.143%	0.263%
probes (~ 100 ng)	~ 40 µL
TOTAL	~ 121 µL

briefly vortex and centrifuge mix
Note: white precipitate will form in Hyb Mix prior to addition of probes. Heat mix @ 65°C for 2 min for precipitate to dissolve. Bring back to room temp before adding probes.

xGen Blocking Oligos

Depending on the library being hybridized, you will need to use the 6nt or 8nt p7 blocker from xGen. The Illumina LT adapters from the 1-12 set have a 6nt variable index area and the 13-24 set have a 8nt variable index area.

TS-p5 and TS-p7 (6nt Index) compatibility

TruSeq LT Adapters 1–12
NEB Adapters 1–12
Bioo Adapters 1–6, 7–12, 13–24
Any single-index adapter with a 6-nucleotide index

TS-p5 and TS-p7 (8nt Index) compatibility

TruSeq LT Adapters 13–27
NEB Adapters 13–27
Bioo Adapters 96-index
Any single-index adapter with an 8-nucleotide index

Dilution of xGen Blockers

IDT recommends resuspending blocking oligos in 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0.
IDT also recommends resuspending blocking oligos to a final concentration of 1 nmole/µL and that we use 1 nmole of the oligos in each capture reaction.
However, from our experiments, we have found we only need 0.1 nmole of blocker per library.

Blockers Mix Recipe - Individual Captures

To make pipetting easier, we make a 1:20 dilution of an aliquot of our stock blocking oligos.

Take 1 µL of each oligo and add it to 19 µL 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0.
This gives a final concentration of 0.05 nmole/µL.

Reagent	x1
Chicken Hybloc (0.5 µg/µL)	2 µL
xGen Universal Blocking Oligo -TS-p5 (0.05 nmole/µL)	2 µL
xGen Universal Blocking Oligo -TS-p7 (0.05 nmole/µL)	2 µL
TOTAL	6 µL

Blockers Mix Recipe - Pooled Captures

To make pipetting easier, let's make a 1:10 dilution of an aliquot of our stock blocking oligos.

Take 1 µL of each oligo and add it to 9 µL 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0.
This gives a final concentration of 0.5 nmole/µL.

Pooling 7 samples

We tried pooling up to 7 samples for a single capture reaction.
xGen Blockers: 0.1 nmole / sample * 7 samples = 0.7 nmole xGen blockers

Blockers Mix Recipe for a 7 sample pooled capture:

Reagent	x1
Chicken Hybloc (1 µg/µL)	5 µL
xGen Universal Blocking Oligo -TS-p5 (0.5 nmole/µL)	1.4 µL
xGen Universal Blocking Oligo -TS-p7 (0.5 nmole/µL)	1.4 µL
TOTAL	7.8 µL

Aliquot 6 µL Blockers Mix to each tube
Add library to Blockers Mix aliquot, mix by pipetting up and down 10X

Amount of capture libraries to add

Want 7-10 µL of total volume of libraries.
May need to evaporate excess liquid to concentrate the libraries (see section 2.7).

3.2 Incubation

Use tube incubators for incubations (vs. thermal-cyclers)

Reaction Profile

Step	Temperature (°C)	Time
1	95	5 min
2	55	5 min
3	55	hold

As summarized above in the reaction profile:
- incubate Blocker Mix + library at 95°C for 5 min
- incubate Blocker Mix + library at 55°C for 5 min
- incubate Hybridization Mix at 55°C for 5 min
- after completion of 5 minutes, leaving tubes in the incubators, quickly add each Hybridization Mix into each Blocker Mix + library
  - ensure cap is screwed tightly on hybridization tube
Quickly transfer tube to tube rotator in pre-heated (55°C) incubator
Snap tube into place and turn on rotator
Close glass door, cover with foil, and tape door shut
- hybridize (incubate at 55°C) for 65-72 hours

Note: 1.5-2 hours before end of hybridization: turn on one tube incubator to 65°C and one to 80°C & prepare Wash Buffer

3.3 Prepare Wash & Binding Buffers

Wash Buffer Recipe:

Reagent	x33
10% Sodium Dodecyl Sulfate (SDS)	400 µL
H2O	39.6 mL
0.1X SSC, 0.1% SDS (= MyBaits Wash Buffer #2)	10 mL
TOTAL	50 mL

vortex thoroughly to mix
heat Wash Buffer aliquots to 65°C for at least 45 min before use

2X Binding Buffer Recipe:

2 M NaCl
10 mM Tris-HCl, pH 7.5
1 mM EDTA
0.5% Tween

It is efficient to make 45-50 mls of the 2X Binding Buffer
From this, prepare 10 mls of 1X Binding Buffer
Bring Dynabeads to room temp and bring aliquot of AmPure beads to room temp

3.4 Dynabead Preparation

To wash Dynabeads:

for each capture reaction, aliquot 50 µL Dynabeads MyOne Streptavidin C1 beads to a 1.5 ml non-stick tube

place on magnetic plate for 2 min until suspension clears
aspirate out supernatant and discard
add 200 µL 1X Binding Buffer to each tube
remove samples from magnetic plate, vortex for 3 seconds, and pulse spin
place on magnetic plate for 2 min until suspension clears
aspirate out supernatant and discard
perform two additional washes with 200 µL 1X Binding Buffer to give a total of three washes
finally, resuspend each washed bead aliquot in 70 µL of 2X Binding Buffer - DO NOT pipette mix. Add buffer, gentle vortex and pulse spin.

3.5 Bead Binding & Washing

Binding of Beads to Probes

heat bead aliquot to 65°C for at least 10 min (use tube incubator)

transfer capture reaction to heated bead aliquot, invert tube to mix
incubate beads + captures at room temp for 30 min on nutator

Bead Washing

quick spin
place beads + captures on magnetic stand for 2 min

remove supernatant
add 500 µL Wash Buffer (heated to 65°C) to the beads, vortex for 3 seconds, and pulse centrifuge
incubate at 55°C for 10 min (in incubator, on rotator)
place on magnetic stand for 1-2 min until suspension clears
remove supernatant
perform two additional washes with 500 µL Wash Buffer for a total of three washes
following the last wash and pelleting on the magnetic stand, remove as much liquid as possible without touching the bead pellet
resuspend in 30 µL 10 mM Tris-HCl, pH 8 (pre-heated to 80°C)
gentle vortex and incubate for 10 min at 80°C, quick spin before magnetizing

3.6 RT-PCR

using HiFi Real-Time PCR Library Amplification Kit (Kapa Biosystems)
- https://www.kapabiosystems.com/product-applications/products/next-generation-sequencing-2/library-amplification/