TITLE: Construction of individuals ddRADseq libraries for macro-algae (Kelp) V.3
AUTHORS: Stéphane Mauger, Komlan Avia

[DESCRIPTION]
This protocol describes a double digested restriction-site associated DNA (ddRADseq) procedure, that is a variation on the original RAD sequencing method (Davey & Blaxter 2011), which is used for de novo SNP discovery and genotyping.

This protocol differs from the original ddRADseq protocol (Peterson et al 2012), in which the samples are pooled just after the ligation to adaptors (i.e. before size selection and PCR). This protocol is an update of the protocol from Claire Daguin Thiebaut et al. (dx.doi.org/10.17504/protocols.io.bv4tn8wn) adapted for macro-algae.

The following protocol is intended for the construction of individual ddRADseq libraries from genomic DNA of various macro-algae samples (Kelp). In the present protocol, we added a genomic DNA purification step to eliminate the inhibitors of PCR and Ligation present in macro-algae (polysaccharides). Moreover, all samples are treated separately until final PCR amplification (Rad Taq enrichment step) performed before pooling.
Despite being slightly more costly and time-consuming in the lab, it allows for fine adjustement of each sample representation in the final library pool ensuring similar number of reads between samples. Finally, we have defined new P1 adapters (barcodes) with variable sequences and variable sizes (6bp to 13bp) to increase the efficiency of the Illumina sequencing.

Briefly, purified genomic DNA from the samples are individually digested with 2 restriction enzymes PstI/HhaI or PstI/MesI (one rare-cutter and one more frequent cutter) then ligated to a barcoded adaptor (among 24 available) at one side, and a single adaptor at the other side, purified with magnetic beads, and PCR-amplified allowing the addition of a Illumina index (among 24 available) for multiplexing a maximum of 576 samples per library. Samples are then pooled in equimolar conditions after visualisation on an agarose gel. Purification and size selection is then performed before final quality control of the library and sequencing.

This protocol has proven its effectiveness in several genetic studies of marco-algae populations.


 


 


 





[BEFORE_START]
1. Prepare all buffers and solutions in advance (see Step 1 to Step 5)
2. If not using Retsch‱ Mixer Mill MM 301 (or equivalente) and Grinding ball for the sample grinding, you can use  Lysing Matrix H tube with FastPrep-24‱ Classic or manual grinding as a last resort.

[STEPS]
SECTION: Solutions and buffers preparations
2. Annealing buffer stock (10x)
Annealing buffer composed 100 mM Tris-HCl, pH8; 500 mM NaCl and 10 mM EDTA 
5 mL
5 mL
1 mL 
39 mL 
 
Homogenize and autoclave.

Store at Room temperature 

  
  

SECTION: Solutions and buffers preparations
1. 5 M sodium chloride solution (NaCl)
29.2 g 
Dissolve the slat in MilliQ water and fill up to 100 mL. 
Autoclave.

Store at Room temperature  

  

SECTION: Preparation of double-stranded barcoded P1 adaptors 4µM
3. Single-stranded oligos NGS grade P1 need to be annealed with their appropriate partner before ligation. We provide sequences for 48 uniquely barcoded adapter P1 oligo pairs (oligos P1_PstI_x.F and P1_PstI_x.R), see the Barcoded_P1_adaptors.xlsx file below.
To create Adapter P1, combine each oligo Forward with its complementary oligo Reverse in a 1:1 ratio in working strength annealing buffer (final buffer concentration 1x) for a total annealed adapter
concentration of 4µM.

In house barcoded P1 adaptors sequences (NGS grade needed) :

  

SECTION: Preparation of double-stranded barcoded P1 adaptors 4µM
3.1. In a PCR plate wells, combine each oligo P1_PstI_x.F with its complementary oligo P1_PstI_x.R :
4 µL
4 µL 
10 µL 
82 µL 

SECTION: Preparation of double-stranded barcoded P1 adaptors 4µM
3.2. 
 

SECTION: Preparation of double-stranded barcoded P1 adaptors 4µM
3.3. The reaction is performed in a thermocycler with the following PCR cycling conditions :

 

Store at 4 °C (or at -20 °C for a long-term storage)

SECTION: Preparation of double-stranded P2 adaptors 40µM
4.2. The reaction is performed in a thermocycler with the following PCR cycling conditions :
 
 

Pool all reaction in a 1.5 mL tube.
Store at 4 °C  (or at -20 °C for a long-term storage).

SECTION: Preparation of double-stranded P2 adaptors 40µM
4.1. In 1.5mL microtube, combine oligo P2_HhaI.F with its complementary oligo P2_HhaI.F (or P2_MseI.F and P2_MseI.R) 
400 µL
400 µL 
100 µL 
100 µL 
 
Then aliquot this volume into 125 µL in each well of a 8- PCR tube strip.

SECTION: Preparation of double-stranded P2 adaptors 40µM
4. Single-stranded oligos NGS grade P2 need to be annealed with their appropriate partner before PCR. We provide sequences for 4 uniquely adapter P2 oligo pairs (oligos P2_HhaI.F and P2_HhaI.R or P2_MseI.F and P2_MseI.R), see the No-Barcoded_P2_adaptors.xlsx file below.
To create Adapter P2, combine each oligo Forward with its complementary oligo Reverse in a 1:1 ratio in working strength annealing buffer (final buffer concentration 1x) for a total annealed adapter
concentration of 40µM.

No-barcoded P2 adaptors sequences (NGS grade needed) :

  

SECTION: Genomic DNA extraction and purification
6. Genomic DNA extraction 
Upon collection, a piece of tissue was cut out from a spot that was free of algal and animal epiphytes and stored in silica gel. Total genomic DNA was extracted from 15 to 20 mg of grinded dry tissue using the Nucleospin 96 plant kit (Macherey-Nagel, Germany).

 

   
SECTION: Genomic DNA extraction and purification
6.1. In a Rack of Tube Strips (consumable of NucleoSpin 96 Plant II kit) add15 mg to 20 mg of each sample with one 3 mm grinding ball stainless steel. Close the Tubes Strips with Cap Strips.

 

  
SECTION: Genomic DNA extraction and purification
6.2. Grind dry tissues using Mixer Mill MM400 Retch using  2 cycles of 2 min at maximum frequency.

 

  
SECTION: Genomic DNA extraction and purification
6.3. The extraction was performed according to the manufacturer's instructions using the PL1 lysis buffer except that we added one wash step with PW1 buffer (2 times PW1 washes in total) and one wash step with PW2 buffer (3 times PW2 washes in total). The extracted DNA was eluted into 120 μL (2 x 60 µL) of the supplied elution buffer.

  

Store at 4 °C  (or at -20 °C for a long-term storage)

  

SECTION: Genomic DNA extraction and purification
7. Genomic DNA purification
The genomic DNA extracts were purified using the NucleoSpin gDNA Clean-up XS, Micro kit for DNA clean up and concentration (Macherey-Nagel, Germany).
The purifications were performed according to the manufacturer's instructions with elution into 30 μL (2 x 15 µL) of the supplied elution buffer.
SECTION: Genomic DNA extraction and purification
7.1. Transferring the 120 µL into 1.5 mL microtubes. Add nuclease free water to fill up to 400 µL.  

SECTION: Genomic DNA extraction and purification
7.2. The purifications were performed according to the manufacturer's instructions with elution into 30 μL (2 x 15 µL) of the supplied elution buffer.

  

SECTION: Genomic DNA extraction and purification
7.3. Removal of residual ethanol and concentration were performed by incubation 15 min at 70 °C 

SECTION: Genomic DNA extraction and purification
7.4. The purified gDNA of each sample was transferred in a 96 wells PCR plate.

Store at 4 °C  (or at -20 °C for a long-term storage)

  

SECTION: Genomic DNA Quality and Quantification
8. Quality Control of Genomic DNA
Optional: load 1 µL  to 3 µL of the Genomic DNA extract on an agarose gel to evaluate its quantity and quality using electrophoresis.

 

SECTION: Genomic DNA Quality and Quantification
9. Quantification of Genomic DNA (Preparation for one 96 wells PCR plate) using PicoGreen‱
Quantify Genomic DNA extract using PicoGreen‱. 

 

SECTION: Genomic DNA Quality and Quantification
9.2. Preparation of 11 mL of 1X TE buffer
In 15 mL 
550 µL 
10.450 mL 
Mix by inverting the tube several times.

SECTION: Genomic DNA Quality and Quantification
9.3. Preparation of DNA solution at 5000 pg/µl (for 3 ranges)
In 0.5 mL 
4 µL 
76 µL 
Mix by inverting the tube several times.

SECTION: Genomic DNA Quality and Quantification
9.5. Pipette 49 µl of 1X TE buffer in the remaining wells.
49 µL

SECTION: Genomic DNA Quality and Quantification
9.6. Pipette 1 µl of the unknown DNA samples in the remaining wells.
1 µL 

SECTION: Genomic DNA Quality and Quantification
9.8. Pipette 50 µL in each well, including the standard and unknown sample wells.

SECTION: Genomic DNA Quality and Quantification
9.11. Plot the measured fluorescent values of the standard samples against their known concentrations and fit a linear curve using linear regression. Make sure that the coefficient of determination (R2) is close to 1 (typically > 0.99). Calculate the DNA concentrations in the unknown samples using the slope and intercept parameters of the linear equation. Output values you obtained are in pg/µl, assuming 1 µl of each sample was used. 
SECTION: Genomic DNA preparation
10. In a PCR plate, put around 100 ngof genomic DNA in a volume of 40 µL (in nuclease free water or Tris-HCl 5mM pH 8.5) for each sample.  If possible, randomize the location of samples in the microplate. Keep a few empty wells for negative controls.
SECTION: Bead purification (96‑well plate format)
13. This protocol can be used to remove contaminants, unligated adapters, enzymes,  buffer  additives,  salts...  and  short  DNA  fragments.  The  method  utilizes  a  single-size  selection  step  :  After  adding  the  appropriate  volume  of  Bead  Suspension  to  the  DNA  sample,  beads  will  bind larger fragments. The supernatant contains smaller fragments and contaminants that are discarded. For most NGS sequencing applications it is optimal to remove all fragments below 100 bp. This can be achieved by using a volume ratio (bead suspension to sample) of 1:1,  which  is  described  in  the  following  protocol.


 

  

SECTION: Bead purification (96‑well plate format)
14. Before starting
Prepare 50 mL of fresh 80% Molecular Biology Grade Ethanol
40 mL 
10 mL 

  


Remove the NucleoMag® NGS Bead Suspension from the fridge. Let for approximately 30 min to bring the bead suspension to Room temperature.
Then, vortex this  Bead Suspension stock solution carefully until homogenized and put in a reagent reservoir.
SECTION: Double digestion
11.1. Vortex all reagents, except enzymes (stored at -20 °C), for approximately 5 s   
Spin down all reagents for approximately 5 s   and placeon ice.
In a microtube, prepare the digestion mix, according to the following table for a total volume of 50 µL:

 

  
  
  
  

SECTION: Double digestion
11. Double digest around  100 ng of high quality genomic DNA with selected restriction enzymes 50 µLreaction volume. Use a digestion buffer appropriate for both enzymes.  Here, we will do the protocol for the PstI and HhaI couple of enzymes but it's same with PstI and MseI couple. Both couple of enzyme woks well for micro-algae but it's possible to test double digestion on few sample to select the best couple of enzymes. The best couple given large smear with size range 100 bp to 1000 pb.

SECTION: Genomic DNA Quality and Quantification
9.10. Place the plate in a plate reader and measure the fluorescence according to the following parameters:


Excitation                   ~480 nm
Emission                    ~520 nm
Integration time            40 s
Lag time                         0 s
Gain                                Optimal
Number of flashes      10
Calculated well             highest standard
Shaking                          5 s


 

 

SECTION: Genomic DNA Quality and Quantification
9.9. Protect the 96-well plate from light and incubate for 5 min at room temperature.

SECTION: Genomic DNA Quality and Quantification
9.7. Prepare PicoGreen‱ work solution
In 10 mL 
25 µL 
4.975 mL 
 Mix and protect from light.
SECTION: Genomic DNA Quality and Quantification
9.4. Preparation of the standard range 0 pg/µl to 1000 pg/µl
Prepare the following standard mixture in 8 0.5 mL 
 
Pipette 50 µL of each standard mixture in the first two columns of the black, sterile, 96-well plate :

 

SECTION: Genomic DNA Quality and Quantification
9.1. Take out all reagents from the fridge and bring them to room temperature.
Take out the DNA samples from the freezer. DNA samples should be slowly thawed on ice


 

 

  

  

SECTION: Bead purification (96‑well plate format)
15. Binding
This step binds DNA fragments 100 bp and larger to the magnetic beads.
Pipette 45 µL of NGS Beads suspension with x8 multichannel pipette and transfer in digestion plate (plate with 45 µL of digested template DNA for each sample), carefully mix by pipetting up and down 10 times. 
Incubate 5 min atRoom temperature 

SECTION: Bead purification (96‑well plate format)
16. Separation
Place the purification plate onto the 96-well magnetic separator.
Wait at least 5 min  until all the beads have been attracted by the magnets or until the liquid appears clear.
The supernatant contains unwanted low molecular weight contaminants and unwanted smaller DNA fragments.
Remove and discard the supernatant (~90 µl) by pipetting.


 


 

  
SECTION: Bead purification (96‑well plate format)
17. 1st wash with 80 % ethanol
Place 80% ethanol in a reagent reservoir.
With a x8 multichannel pipette, dispense200 µL  of 80% ethanol into the purification plate without disturbing the bead pellet. 
Incubate the purification plate at room temperature for at least 30 s
Carefully and completely remove and discard ethanol by pipetting.
SECTION: Bead purification (96‑well plate format)
19. Dry the beads
Let the purification plate on the magnetic separator and incubate at Room temperature for maximum 5 min in order to allow the remaining traces of ethanol to evaporate.

 

SECTION: Bead purification (96‑well plate format)
18. 2nd wash with 80 % ethanol
With a x8 multichannel pipette, dispense200 µL  of 80% ethanol into the purification plate without disturbing the bead pellet. 
Incubate the purification plate at room temperature for at least 30 s
Carefully and completely remove and discard ethanol by pipetting.
SECTION: Check digestion on an agarose gel
12. Check the efficiency of the digestion by electrophoresis of 5 µL of digested DNA  in a 1.5 % agarose gel (standard quality). High molecular weigh DNA should no longer be visible.

 

SECTION: Adaptor ligation
21. For each sample of one line of the digested purified plate (with 35 µL of digested purified template DNA) add  5 µL of double-stranded barcoded P1 adaptors at 4 µM. Use one  double-stranded barcoded P1 adaptors per line.

 

 


 

SECTION: Adaptor ligation
22. Vortex all reagents, except enzymes (stored at -20 °C), for approximately 5 s   
Spin down all reagents for approximately 5 s   and placeon ice.
In a microtube, prepare the ligation mix,  according to the following table for a total volume of 60 µL:
 
 
 
SECTION: Double digestion
11.2. Vortex the master mix and spin down.
Aliquot 125 µL   of the digestion master mix in each well of a 8-PCR tube strip.
In the DNA plate (containing 40 µL per well), add10 µL  of digestion master mix with a x8 multichannel pipette and mix by pipetting, seal PCR plate and spin down.

Incubate at 37 °C 5 s 
Then store at 4 °C 

SECTION: Adaptor ligation
23. Vortex the master mix and spin down.
Aliquot 125 µL   of the ligation master mix in each well of two 8-PCR tube strip.
In the digested purified plate (containing 35 µL of digested purified template DNA and 5 µLof  barcoded P1 adaptors ), add20 µL  of ligation master mix with a x8 multichannel pipette and mix by pipetting, seal PCR plate and spin down.

Incubate at 16 °C 5 s 
Then store at 4 °C or at  -20 °C if not performing the bead purification the day after.

SECTION: Bead purification (96‑well plate format)
30. Elute DNA fragments
Take the purification plate from the magnetic stand,  and add 40 µL  of nuclease-free water with a x8 multichannel pipette to resuspend the bead pellet by pipetting up and down 10 times.
Incubate the purification plate at Room temperature for 5 min  .
Separate the magnetic beads against the side of the wells by placing the 96-well plate on the  magnetic separator.
Wait at least 5 min   until all the beads have been attracted by the magnets or until the liquid appears clear.
Transfer 35 µL  of the supernatant containing the adaptor-ligated purified template DNA to a new 96‑well plate. Be careful to avoid pipeting beads during this step. 

Seal the plate and store at 4 °C  (or store -20 °C for a long-term storage) until PCR amplification.
SECTION: Bead purification (96‑well plate format)
24. Before starting
Prepare 50 mL of fresh 80% Molecular Biology Grade Ethanol
40 mL 
10 mL 

  


Remove the NucleoMag® NGS Bead Suspension from the fridge. Let for approximately 30 min to bring the bead suspension to Room temperature.
Then, vortex this  Bead Suspension stock solution carefully until homogenized and put in a reagent reservoir.

  

SECTION: Bead purification (96‑well plate format)
25. Binding
This step binds DNA fragments 100 bp and larger to the magnetic beads.
Pipette 60 µL of NGS Beads suspension with x8 multichannel pipette and transfer in adaptor-ligated plate (plate with 60 µL of digested and adaptor-ligated template DNA for each sample), carefully mix by pipetting up and down 10 times. 
Incubate 5 min atRoom temperature 

SECTION: Bead purification (96‑well plate format)
26. Separation
Place the purification plate onto the 96-well magnetic separator.
Wait at least 5 min  until all the beads have been attracted by the magnets or until the liquid appears clear.
The supernatant contains unwanted low molecular weight contaminants and unwanted smaller DNA fragments.
Remove and discard the supernatant (~120 µl) by pipetting.


 


 

  
SECTION: Bead purification (96‑well plate format)
27. 1st wash with 80 % ethanol
Place 80% ethanol in a reagent reservoir.
With a x8 multichannel pipette, dispense200 µL  of 80% ethanol into the purification plate without disturbing the bead pellet. 
Incubate the purification plate at room temperature for at least 30 s
Carefully and completely remove and discard ethanol by pipetting.
SECTION: Bead purification (96‑well plate format)
28. 2nd wash with 80 % ethanol
With a x8 multichannel pipette, dispense200 µL  of 80% ethanol into the purification plate without disturbing the bead pellet. 
Incubate the purification plate at room temperature for at least 30 s
Carefully and completely remove and discard ethanol by pipetting.
SECTION: Bead purification (96‑well plate format)
29. Dry the beads
Let the purification plate on the magnetic separator and incubate at Room temperature for maximum 5 min in order to allow the remaining traces of ethanol to evaporate.

 

SECTION: Bead purification (96‑well plate format)
20. Elute DNA fragments
Take the purification plate from the magnetic stand,  and add 40 µL  of nuclease-free water with a x8 multichannel pipette to resuspend the bead pellet by pipetting up and down 10 times.
Incubate the purification plate at Room temperature for 5 min  .
Separate the magnetic beads against the side of the wells by placing the 96-well plate on the magnetic separator.
Wait at least 5 min   until all the beads have been attracted by the magnets or until the liquid appears clear.
Transfer 35 µL  of the supernatant containing the digested purified template DNA to a new 96‑well plate. Be careful to avoid pipeting beads during this step. 

Seal the plate and store at 4 °C  (or store -20 °C for a long-term storage) until adaptor ligation.

SECTION: Rad Tag enrichment (PCR)
31. PCR amplification to generate Illumina sequencing indexed libraries :
In this PCR, Illumina indexed primers are incorporated in order to produce fragments compatible with Illumina sequencing, and to insert an index allowing multiplexing of barcoded samples. This index will be read during the sequencing run.
This PCR is expected to have a homogenizing effect. Primers are thus included in limiting quantity, in order to produce equalized amounts of PCR fragments among samples. The number of cycles is limited to a maximum of 15 (optimal with 12). After those cycles, a final PCR cycle is then performed after addition of primers in large excess.

The Reaction mixture for a total volume of 40 µL is :

 

We need to prepare one PCR mixture per index, i.e. 12 PCR mixtures for one plate.


 


  

SECTION: Rad Tag enrichment (PCR)
32. First PCR mix preparation (with primers in limiting quantity)
Defreeze and vortex all reagents, except enzymes (stored at -20°C), for approximately5 s  
 Spin down all reagents for approximately5 s   and place on ice.

In 12 0.5 mL microtubes, prepare the 1st mix according to the following table (one mix per column) : 
 
 

Vortex mix all reagents in the mix and spin down.
In a new PCR plate, dispense 30 µL  of 1st mix in each column.

SECTION: Rad Tag enrichment (PCR)
33. DNA and mix combination 
Spin down the adaptor-ligated purified template DNA plate.
With a multichannel pipette, transfer 10 µL   of adaptor-ligated purified template DNA into the PCR plate and mix by pipetting.
Finally, aliquot the 40 µL of the total mix by dispensing 20 µL  into 1 additional new empty PCR plates.
Seal the 2 PCR plates and spin down. 


The 2 PCR will be performed in parallel in 2 different thermal cyclers, in order to reduce the PCR bias.

 

After PCR, pool back the 2 PCR plates into a single plate with a multichannel 

SECTION: Rad Tag enrichment (PCR)
34. Final cycle (with primers in large excess)
In a 12-tube PCR strip, prepare the 2nd mix according to the following table (one mix per column) :

 
 
Mix all reagents by pipetting and spin down.

Dispense 4 µL of final cycle mix in each line of the PCR plate with a 12 multichannel pipette.
Seal the PCR plate and spin down.

In a thermocycler, run the final cycle as follows :

 

After PCR, place the plate at4 °C (or -20 °C for a long-term storage).

SECTION: Preparation of Illumina indexed primers mix (5µM)
5. In 24 1.5 mL microtubes, combine each of the 24 Illumina indexed reverse primers ILLPCR2_ind01 to ILLPCR2_ind27  (no primer numbers ind17, ind24 and ind26) with the Illumina no-indexed forward primer ILLPRC1, see the Illumina_indexed_primers.xlsx file below.
5 µL ILLPCR1 oligo forward (100µM)
5 µL ILLPCR2 oligo reverse (100µM) ind01 to ind27 (one per tube)
90 µL nuclease free water and mix by pipetting

Store at 4 °C (or at -20 °C for a long-term storage)

Ilumina indexed primers sequences (NGS grade needed) :

  

SECTION: Check PCR on an agarose gel
35. Check the efficiency of the PCR by electrophoresis of 5 µL of digested DNA  in a 1.5 % agarose gel (standard quality).


 


SECTION: Bead purification (microtube format)
37. Before starting
Prepare 10 mLof fresh 80% Molecular Biology Grade Ethanol
8 mL 
2 mL 

  


Remove the NucleoMag® NGS Bead Suspension from the fridge. Let for approximately 30 min to bring the bead suspension to Room temperature.
Then, vortex this  Bead Suspension stock solution carefully until homogenized and put in a reagent reservoir.

  

SECTION: Bead purification (microtube format)
38. Binding
This step binds DNA fragments 100 bp and larger to the magnetic beads.
Pipette a volume of NGS Beads suspension to have a ratio 1:1 with the sample pooling volume, and transfer in the pooling sample tube(s). 
Carefully mix by pipetting up and down 10 times. 
Incubate 5 min atRoom temperature 

SECTION: Bead purification (microtube format)
39. Separation
Place the purification tube(s) onto the magnetic microtube stand.
Wait at least 5 min  until all the beads have been attracted by the magnets or until the liquid appears clear.
The supernatant contains unwanted low molecular weight contaminants and unwanted smaller DNA fragments.
Remove and discard the supernatant by pipetting.


 


  

SECTION: Bead purification (microtube format)
43. Elute DNA fragments
Take the purification tube(s) from the magnetic stand,  and add 100 µL  of nuclease-free water with a  pipette to resuspend the bead pellet by pipetting up and down 10 times.
Incubate the purification tube(s) at Room temperature for 5 min  .
Separate the magnetic beads against the side of the tube by placing the tube(s) on the  magnetic separator.
Wait at least 5 min   until all the beads have been attracted by the magnets or until the liquid appears clear.
Transfer 90 µL  of the supernatant in a new(s) low binding 1.5 mL microtube(s). Be careful to avoid pipeting beads during this step. 

Store at 4 °C  (or store -20 °C for a long-term storage) until size selection.
SECTION: Bead purification (microtube format)
40. 1st wash with 80 % ethanol
Dispense1 mL  of 80% ethanol into the purification tube(s) without disturbing the bead pellet. 
Incubate the purification tube(s) at room temperature for at least 30 s
Carefully and completely remove and discard ethanol by pipetting.
SECTION: Bead purification (microtube format)
41. 2nd wash with 80 % ethanol
Dispense1 mL  of 80% ethanol into the purification tube(s) without disturbing the bead pellet. 
Incubate the purification tube(s) at room temperature for at least 30 s
Carefully and completely remove and discard ethanol by pipetting.
SECTION: Bead purification (microtube format)
42. Dry the beads
Let the purification tube(s) on the magnetic separator and incubate at Room temperature for maximum 5 min in order to allow the remaining traces of ethanol to evaporate.

 

SECTION: Sample pooling (equimolar)
36. Each barcoded and indexed individual can now be pooled in a single tube, in equimolar conditions.

After the normalizing PCR, all smears should have similar intensity on the agarose gel. In this case, pool 5 µL of all individuals in a single low binding 1.5 mL microtube. 

If not, normalization can be made at this step. For this, roughly estimate the concentration of fragments from the gel picture, and pools accordingly.  It can be efficient to make intermediate pools for example, one pool for the low,  one for the medium, and another one for the high intensity samples in 3 low binding 1.5 mLmicrotubes .

Vortex mix and spin down.

Store at4 °C (or -20 °C for a long-term storage) until bead purification.

SECTION: Size selection with sage science Pippin-Prep
45. Perform the size selection of fragments between 300 and 800 bp using a 1,5% DF marker K agarose gel cassette, according to the Pippin prep manufacturer's instructions : 

  


 


SECTION: Intermediate pools quantification and final pooling (Optional)
44. If you made intermediate pools (low, medium and high intensity on agarose gel), estimate the double strand DNA concentration in the pools by fluorimetry with a Qubit equipment.

  

Pool in equimolar concentration the 3 intermediate pools in a single low binding 1.5 mLmicrotube with a minimum concentration of 20 nanomolar (nM) and minimum volume of 30 µL.  

Vortex mix and spin down.

Store at4 °C (or -20 °C for a long-term storage) until size selection.
SECTION: Quality control of the libraries
46. Control the quality of the library with a Bioanalyzer (Agilent) (or equivalent equipment) in a High Sensitivity DNA chip. Dilute your pool 1:2 or more and load 1µl of the pool before and after size selection, according to the manufacturer's instructions :

  


 


 


SECTION: Quality control of the libraries
47. Fluorimetric estimation of the dsDNA concentration in the library.
Perform a quick estimation of the DNA concentration with a fluorimetric assay, in a QubitTM apparatus or equivalent, with the Qubit ds 1X DNA HS assay kit, according to the manufacturer's instructions:

  

SECTION: Quality control of the libraries
48. qPCR quantification
In the case you need an accurate estimation of the DNA concentration in your library, perform a qPCR quantification with the NEBNext Library Quant Kit for Illumina, or equivalent, which uses P5 and P7 illumina primers to target the double stranded DNA fragments in the library. Follows the kit's user guide and perform your quantitative qPCR in a qPCR thermocycler (e.g. LightCycler 480,  Roche).

  

Contrarily to the fluorimetric method (Qubit), the qPCR estimation will only consider dsDNA fragments starting with P5 and ending with P7 illumina sequences, that will be effectively amplified onto the flowcell of the Illumina sequencer.
SECTION: Quality control of the libraries
49. Library ready for sequencing
The library is now ready for sequencing in single read or paired-end 150 bases in an Illumina sequencer, with one index read.
SECTION: Quality control of the libraries
48.1. Suggestions to prepare library dilutions for qPCR
In 0.5 mL  low binding microtube, prepare 1:1 000 dilution of library with buffer supplied in the qPCR kit. Then, prepare the 3 library dilutions (1:10 000 to 1:30 000) to be used on triplicate for qPCR analysis.
1:100 : 1 µL of library + 99 µL of 1X buffer
1:1 000 : 10 µL of library + 90 µL of 1X buffer

1:10 000 : 20 µL of 1:1 000 dilution + 180 µL of 1X buffer 
1:20 000 : 50 µL of 1:10 000 dilution + 50 µL of 1X buffer 
1:30 000 : 50 µL of 1:10 000 dilution + 100 µL of 1X buffer 

You should get more than 10 nM, that is the library concentration usually required by the sequencing platform facilities. 
Use the average size of the library size range as estimated from the Bioanalyzer profile to convert DNA concentration from nM to ng/µL using the attach file below :