TITLE: A FAIR protocol of the Best-RAD sequencing approach
AUTHORS: Laure Benoit, Sabine Nidelet, Emeline Charbonnel, Marie-Pierre Chapuis

[DESCRIPTION]
The protocol is based on the Best-RAD sequencing  approach developed in Ali et al. (2016) that allows two main improvements of the RAD-sequencing approach developed by Baird et al. (2008) : (1) a lower rate of PCR duplicates generated during the final enrichment and (2) an increase of the multiplexing capacity by a factor equal to the number of well barcode available (for a brief description, see Figure 1). We developed this protocol on individual wild samples of the Oriental fruit fly, Bactrocera dorsalis. We evaluated the library quality not only on a high amount of target DNA fragments but also on low amounts of PCR duplicates, chimeric fragments, and adaptor residues. To this aim, we tested and validated the following critical parameters : the quality and quantity of the DNA input, the ratio of the AMpure purifications and the number of PCR cycles of the final amplification. Consecutive recommendations and expectations (e.g., DNA concentration or fragment size) are indicated in notes throughout the protocol. Furthermore, special care has been taken to ensure the precision of each step (i.e. volumes, quantities, duration, materials with supplier references). Consequently, this protocol follows the FAIR principle and could be useful for an easy implementation of the Best-RAD sequencing approach in other laboratories and biological models.

 


[BEFORE_START]
DNA quality control
Beforehand, DNA quality should be checked with an electrophoresis on an agarose gel.

Measurement of DNA quantity
Beforehand, the amount of genomic DNA should be determined with a fluorescent method (as Qubit assay) in order to be normalized. 

Sample metadata
Prepare a metadata file containing for each sample, a unique ID and the combination of the three barcodes used. This allows to avoid switching barcodes during the sample pooling and is required for sequence read demultiplexing.

[GUIDELINES]
Enzymes
Both PstI and SbfI enzymes are compatible with this protocol.

DNA quality
Wherever possible, select input DNA samples of a molecular weight of at least 5kb. A more degraded DNA sample could be lost and should be avoided.  Note that we worked on DNA extracted using a 96-Well Plate Animal Genomic DNA Miniprep Kit (Biobasic, Toronto, Canada, Cat.#: BS437) according to the manufacturer’s protocol, except that lysis was made over-night  and followed by an RNA degradation step using 4µL of RNase A (Qiagen, 100 mg/ml, Cat.#: 19101) during 2 min at room temperature.

Sample multiplexing
The protocol is designed to process samples in batches of 96 (i.e. to work in a 96-well plate format), but it is possible to work with fewer samples (e.g. batches of 24 samples).
Each sample must be associated to a unique ID and three multiplexing barcodes. These barcodes are :
(1) a well biotinylated barcode as defined by Ali et al. (2016) that is unique to a sample within a batch (the list of barcode sequences is available in the Supplementary data FileS1; "New RAD" table). 
(2) the two P1 and P2 plate barcodes added to the Illumina adapters that are, in combination, unique to a batch (the list of sequences we used is available at the materials section). Any index compatible with illumina technology could be used, such as NEBNext Multiplex Oligos for Illumina (Cat.#: E7395S).

Control samples
Including one negative control (i.e. empty well) randomly located in each plate allows to easily identify possible technical errors (e.g. 96-well plate switch).
Including technical replicates (i.e. include twice the same DNA sample with different barcodes) allows to estimate genotyping error rate.

[STEPS]
SECTION: Part II - Selection of fragments carrying the restriction site
5. Preparation of Dynabead M-280 magnetic streptavidin beads

Since streptavidin beads adhere to tips, re-suspend by vortexing and not by pipetting.

Incubate the streptavidin beads at room temperature for at least  30 min before using it.


For each pool, prepare 1 mL  2X Binding and Wash buffer (B&W 2X): 
10 millimolar (mM) 
1 millimolar (mM) 
2 Molarity (M) 

For each pool, transfer 30 µL of Dynabeads into a new 1.5mL low bind tube.
Place the tube on a magnetic holder and discard the supernatant.
Without removing the tube from the magnetic holder, add 100 µL
Remove the tube from the magnetic holder, vortex 30 s, centrifuge briefly.
Place the tube on the magnetic holder for 1 min and discard the supernatant.
Repeat steps 3 to 5 twice for a total of 3 washes.
Re-suspend beads in 100 µL.
SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
1. Genomic DNA enzymatic restriction
SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
2. Ligation of well barcodes

SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
4. Shearing

Maintain tubes at 4°C if shearing is planned within 24 hours or freeze.

SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
1.2. Into a 1.5 ml low bind tube on a cold block, prepare the master mix of enzymatic restriction (vols for 1 rxn) :

0.55 µL  
1.2 µL 
0.25 µL 


Gently vortex and centrifuge the mix.
Using a new plate on a cold block, a Multipette and a 0.5mL Combitip, distribute 2 µL of mix into each well.
Using P10 multichannel pipet, add 10 µL of normalized DNA.
Close the plate with self-adhesive film.
Vortex and centrifuge.
SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
1.3. 
Apply the following restriction program using a thermocycler (heated lid on):

37 °C
80 °C 
10 °C 

SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
2.1. Into a 1.5 ml low bind tube, prepare the ligation master mix (vols for 1 rxn) :


1.19 µL  
0.4 µL 
0.16 µL 
0.25 µL 


Gently vortex the mix and centrifuge.
Using a new plate on a cold block, a Multipette and a 0.5mL Combitip, distribute 2 µL of mix into each well.
Using P10 multichannel pipet, add 12 µL of digested DNA
Using P10 multichannel pipet, add 2 µL of well barcodes (0.05µM if SbfI digestion or 1µM if PstI) according to the plate plan.
Close the plate with self-adhesive film
Gently vortex and centrifuge
 
SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
2.2. Apply the following ligation program using a thermocycler (heated lid on):


22 °C
65 °C (decrease the temperature slowly to 65 at 25°C, e.g. 2.7°C/min during 15min using the minimum ramp speed)
25 °C 

SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
4.1. 

Briefly vortex and centrifuge the tubes (maximum 12 tubes in parallel, i.e. 12 batches of 96 samples)
Check that there are no bubbles and place the tubes in the rotor (12 tubes of 0.5mL).
Place the rotor in the bath.
Run the following shearing program:
Four cycles of :
30 s 
90 s 

Repeat the four previous steps one more time (for a total of 8 shearing cycles or a different total number of cycles, see note 4.2).
SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
4.2. Quality and quantity control (optional)

Run 1 µL of each pool on an Agilent High Sensitivity (HS) chip (following manufacturer protocol).
 

Run 1 µL of each pool on a Qubit High Sensitivity (HS) assay (following manufacturer protocol).
 

SECTION: Part II - Selection of fragments carrying the restriction site
6. Binding DNA to beads
SECTION: Part II - Selection of fragments carrying the restriction site
7. Release DNA from streptavidin beads

Re-suspend the beads in 100 µL.
Remove the tube from the magnetic holder, vortex gently and centrifuge briefly
Place the tube on the magnetic holder for 1 min and discard the supernatant.
Repeat step 1 to 3 for a total of 2 washes.
Re-suspend the beads in 40 µL.
Remove the tube from the magnetic holder, vortex gently and centrifuge briefly.
Add 2 µL. 
Vortex gently.
Run the following release program in a thermomixer:

37 °C
  

Vortex gently and centrifuge briefly.
Place the tube on the magnetic rack for 1 min.
Transfer 40 µL  supernatant in a 1.5mL low bind tube.

SECTION: Part II - Selection of fragments carrying the restriction site
8. Purification on AMpure beads  (1X)

Incubate AMpure beads at room temperature at least 30 min. 
Prepare 70% ethanol  :
    For one pool: 133 µL +467 µL.


Add 40 µL of AMpure beads to each pool.
Incubate5 min at room temperature
Place on a magnetic holder for 5 min.
Without removing the tube from the magnetic holder, remove supernatant, add 200 µL, incubate 30 s and discard the supernatant.
Repeat step 5 one time
Let dry on the magnetic support for 8 min.
Re-suspend in 51 µL(or 52µL for the optional step 9)
Remove from the magnetic holder and vortex gently 
Incubate 2 min at room temperature
Centrifuge briefly and place on magnetic holder for 5 min.
Transfer 50 µL(or 51µL for the optional step 9) to a 0.2mL PCR tube.
Vortex and centrifuge.
SECTION: Part III - Library construction with NEBNext Ultra II kit
10. 
 
NEBNext End Prep

Directly into the 50 µL from step 8, add :

3 µL  
7 µL 

Vortex gently and centrifuge briefly (It is important to mix well. The presence of a small amount of bubbles will not interfere with performance)
Place in a thermocycler, with the heated lid set to ≥ 75°C
Run the following program:

20 °C
65 °C 
4 °C 

Warning: move to the next step immediately.

SECTION: Part II - Selection of fragments carrying the restriction site
9. Quantity control (optional)


Run 1 µL of each pool on a Qubit HS assay (following manufacturer protocol).
 

SECTION: Part III - Library construction with NEBNext Ultra II kit
11. Plate barcode adapter Ligation


Directly into the 60 µL from step 10, on a cold block, add in order:

30 µL 
1 µL 
1.25 µL 1.5 micromolar (µM) 
1.25 µL 1.5 micromolar (µM) 

Vortex gently and centrifuge briefly (It is important to mix well. The presence of a small amount of bubbles will not interfere with performance)
Place in a thermocycler, with the heated lid off
Run the following program:

20 °C
10 °C 

Samples can be stored overnight at –20°C.
SECTION: Part III - Library construction with NEBNext Ultra II kit
12. Cleanup of Adaptor-ligated DNA (ratio 0.65X)

 

Incubate AMpure beads at room temperature at least 30 min. 
Prepare 70% ethanol  :
    For one pool: 133 µL +467 µL.

Transfer 93.5 µL from step 11 in a 1.5mL low-bind tube.
Add 61 µL
Incubate5 min at room temperature
Place on magnetic holder for 5 min.
Without removing the tube from the magnetic holder, remove supernatant, add 200 µL, incubate 30 s and discard the supernatant.
Repeat step 5 one time.
Let dry on the magnetic support for 8 min.
Re-suspend in 51 µL(52µL for the optional step 13).
Remove from the magnetic holder and vortex gently.
Incubate 2 min at room temperature.
Centrifuge briefly and place on magnetic holder for 5 min.
Transfer 17 µL (18µL for the optional step 13) to a 0.2mL PCR tube.
Vortex and centrifuge.
SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
1.1. In a 96-well microplate, normalize the DNA concentration of each DNA sample in a final volume of 10µL using EB buffer.


 

SECTION: Part III - Library construction with NEBNext Ultra II kit
14. Final PCR enrichment


 

Into a PCR tube, prepare PCR master mix (vols for 1 rxn) :
25 µL 
5 µL 10 micromolar (µM)
5 µL 10 micromolar (µM)

15 µL 

Vortex gently and centrifuge briefly 
Place in a thermocycler, with the heated lid on, and run the following program:

98 °C

6-12 cycles of :
98 °C 
65 °C 

65 °C 
10 °C 
SECTION: Part III - Library construction with NEBNext Ultra II kit
15. Cleanup of amplified library (ratio 0.65X)


 
Incubate AMpure beads at room temperature at least 30 min. 
Prepare 70% ethanol  :
    For one pool: 133 µL +467 µL.

Verify the 50 µL from step 13 and transfert in a new low bind tube.
Add 32.5 µL
Place on a magnetic holder for 5 min.
Without removing the tube from the magnetic holder, remove supernatant, add 200 µL, incubate 30 s and discard the supernatant.
Repeat step 4 one time
Let dry on the magnetic support for 8 min.
Re-suspend in 22 µL
Remove from the magnetic holder and vortex gently 
Incubate 2 min at room temperature
Centrifuge briefly and place on magnetic holder for 5 min.
Transfer 21 µL to a 0.2mL PCR tube.
Vortex and centrifuge.
SECTION: Part III - Library construction with NEBNext Ultra II kit
16. Final quality controls

Run 1 µL of each library on a Qubit HS assay (following manufacturer protocol).
 

Run 1 µL of each library on an Agilent HS chip (following manufacturer protocol).
 

Run 2 µL of each library on a Kapa assay (following manufacturer protocol).
 



SECTION: Part III - Library construction with NEBNext Ultra II kit
13. Quantity control (optional)


Run 1 µL of each pool on a Qubit HS assay (following manufacturer protocol).
 

SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
3.2. Purification and concentration on AMpure beads (ratio 1X)

Incubate AMpure beads at room temperature at least 30 min. 
Prepare 70% ethanol  :
    For one pool: 954 µL +2000 µL.


Measure the exact volume of the pool
Add an equivalent volume of AMpure beads and vortex
Incubate5 min at room temperature
Place on a magnetic holder for 5 min.
Without removing the tube from the magnetic holder, remove supernatant, add 1000 µL, incubate 30 s and discard the supernatant.
Repeat step 5 one time
Let dry on the magnetic holder for 8 min.
Re-suspend in 100 µL (or in 102µL to do the optional 4.2 step)
Remove from the magnetic holder and vortex gently 
Incubate 2 min at room temperature
Centrifuge briefly and place on a magnetic holder for 5 min.
Transfer 100 µL( or 102µL for the optional 4.2 step) to Diagenode tubes for shearing
Vortex and centrifuge.

SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
3. Pooling, purification and concentration
SECTION: Part I - Genomic DNA restriction, well barcode ligation, pooling and shearing
3.1. Sample pooling

 
Using a multichannel pipet, pool each barcoded sample of a same batch and a same row of a plate in a 0.2mL 8-tubes PCR strip.
Transfer all the samples of a batch, i.e. all the volume of the 8-tubes PCR strip, in a 1.5mL low-bind tube.
SECTION: Part II - Selection of fragments carrying the restriction site
6.1. Add shared DNA (~100µL) to the prepared beads.
Briefly vortex and centrifuge the tubes.
Apply the following binding program in a thermomixer:

22 °C
  
SECTION: Part II - Selection of fragments carrying the restriction site
6.2. For each pool:
Prepare B&W 1X: 250 µL + 250 µL. Incubate at Room temperature 
Pepare B&W 1X: 175 µL + 175 µL. Incubate at 56 °C  in a thermomixer.
Prepare NEB buffer 4 1X: 30 µL + 270 µL. Incubate at Room temperature.
SECTION: Part II - Selection of fragments carrying the restriction site
6.3. Centrifuge briefly and transfer each pool in a 1.5mL low bind tube. 
Place tubes on a magnetic holder for 1 min and discard the supernatant.
Re-suspend the beads with 150 µL at Room temperature.
Vortex gently and centrifuge briefly.
Place the tube on the magnetic holder for 1 min and discard the supernatant.
Repeat steps 3 to 5 twice with B&W 1X buffer at Room temperature for a total of 3 washes.
Repeat steps 3 to 5 twice more with buffer B&W 1X at 56 °C for a total of 5 washes.