TITLE: Construction of individual ddRAD libraries
AUTHORS: Claire Daguin Thiebaut, Stephanie Ruault, Charlotte Roby, Thomas Broquet, Frédérique Viard, Alan Brelsford

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes a double digested restriction-site associated DNA (ddRADseq) procedure, that is a variation on the original RAD sequencing method (</span><a href="https://doi.org/10.1093/bfgp/elq031" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Davey & Blaxter 2011</span></a><span>), which is used for </span><span style = "font-style:italic;">de novo</span><span> SNP discovery and genotyping.</span></div><br/><div class = "text-block"><span>This protocol differs from the original ddRADseq protocol (</span><a href="https://doi.org/10.1371/journal.pone.0037135" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Peterson et al 2012</span></a><span>), in which the samples are pooled just after the ligation to adaptors (i.e. before size selection and PCR). </span></div><div class = "text-block">The present ddRAD protocol as been slightly adapted from Alan Brelsford's protocol published in the supplementary material of this paper:</div><br/><div class = "text-block"><span>Brelsford, A., Dufresnes, C. & Perrin, N. 2016. High-density sex-specific linkage maps of a European tree frog (</span><span style = "font-style:italic;">Hyla arborea</span><span>) identify the sex chromosome without information on offspring sex. </span><span style = "font-style:italic;">Heredity </span><span style = "font-weight:bold;">116, </span><span>177–181 (2016). </span><a href="https://www.nature.com/articles/hdy201583/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1038/hdy.2015.83</span></a><span></span></div><br/><div class = "text-block">In the present protocol, all samples are treated separately, in a microplate, until final PCR amplification 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 sequencing reads per sample in the final dataset. </div><br/><div class = "text-block">Briefly, genomic DNA from the samples are individually digested with 2 restriction enzymes (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 12 available) for multiplexing a maximum of 288 sample 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.</div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/eeuaiutw.jpg" /></div><br/><br/><br/><br/><br/><br/><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/eeue2xcn.jpg" /></div><br/></div>

[STEPS]
?. [Preparation of double-stranded barcoded  P1 adaptors 1µM]
In a PCR plate wells, combine each oligo 1.1 with its complementary oligo 1.2 : adaptor P1-1  10µM  adaptor P1-2   10µM annealing buffer 10X (100 mM Tris-HCl, pH 8, 500 mM NaCl, 10 mM EDTA Na2) nuclease free waterSeal the plate with a thermoseal film and incubate in a thermocycler or microplate incubator at   for , and then cool at a rate of 0.1°C / s down to 20°C. Store at 4°C (or at -20°C for a long-term storage)
5 µl
5 µl
5 µl
35 µl
95 °C
?. [Preparation of double-stranded barcoded  P1 adaptors 1µM]
ABCDEFGHIJKL125917221018331119442620551321661422771523881624
ABCDEFGHIJKL125917221018331119442620551321661422771523881624
?. [Preparation of double-stranded P2 Adaptor P2_Mse 10µM]
in a microtube, combine oligo 2.1 with its complementary oligo 2.2  P2-1  100µM P2-2   100µM annealing buffer 10X  (100 mM Tris-HCl, pH 8, 500 mM NaCl, 10 mM EDTA Na2) nuclease free water and mix by pipettingThen aliquot this volume into   in each well of a 8- PCR tube strip.In a thermocyler or microplate incubator, incubate at   for , and then cool at a rate of 0.1°C / s down to 20°C. Store at 4°C (or -20°C for a long-term storage)
80 µl
80 µl
80 µl
560 µl
100 µl
95 °C
?. [Genomic DNA preparation]
In a PCR plate, put around 50-100ng of genomic DNA in a volume of 30µl (in nuclease free water or Tris-HCl 5mM pH 8.5) for each sample. See guidelines for more details. Ideally, DNA should be free from RNA. For this, include a RNAse treatment in the DNA extraction procedure. If possible, randomize the location of samples in the microplate. Keep a few empty wells for negative controls.
?. [Double digestion]
Thaw and vortex all reagents, except enzymes (stored at -20°C), for approximately Spin down all reagents for approximately  and place on ice.In a microtube, prepare the master mix,  according to the following table  for a total volume of 35μL:  ABCDE1concentrationfinal quantityN=1N=104 (1 plate)2genomic DNA50-200ng303Cutsmart buffer10X1X3.53644Enzyme 1 Pst HF20u/µL8U0.441.65Enzyme 2 MseI10u/µl2U0.220.86H2O  0.993.67TOTAL  35 Vortex mix the master mix and spin down.Aliquot  of the master mix in each well of a 8-PCR tube strip.In the DNA plate (containing 30µL per well), add  of Master mix with a multichannel pipette and mix by pipetting, seal PCR plate and spin down.Incubate at then   in order to inactivate enzymes
ABCDE1concentrationfinal quantityN=1N=104 (1 plate)2genomic DNA50-200ng303Cutsmart buffer10X1X3.53644Enzyme 1 Pst HF20u/µL8U0.441.65Enzyme 2 MseI10u/µl2U0.220.86H2O  0.993.67TOTAL  35
65 µl
5 µl
37 °C
65 °C
4 °C
?. [Check digestion on an agarose gel]
Check the efficiency of the digestion by electrophoresis of 5µl of digested DNA  in a 1.5 % agarose gel (standard quality). High molecular weigh DNA should no longer be visible.
?. [Adaptor ligation]
Vortex mix all reagents, except enzymes (stored at -20°C), for approximately Spin down all reagents for approximately  and place on ice.Setup the ligation reaction according to the following table (total volume of 40μL):  AB1N=1 / µl2digest DNA303ds barcoded adaptor P1 1µM2.84ligation mix (see below)7.2 In a microtube, prepare the ligation mix according to the following table :  ABCDE1concentration stockfinal concentrationvol 1x µlVol µl plate2ds nonbarcoded adaptor P210µM 1.8187.23Cutsmart buffer10X1X11044ATP10mM1 mM44165T4 DNA ligase400u/µl0.441.66TOTAL&nbsp; 7.2
AB1N=1 / µl2digest DNA303ds barcoded adaptor P1 1µM2.84ligation mix (see below)7.2
ABCDE1concentration stockfinal concentrationvol 1x µlVol µl plate2ds nonbarcoded adaptor P210µM 1.8187.23Cutsmart buffer10X1X11044ATP10mM1 mM44165T4 DNA ligase400u/µl0.441.66TOTAL&nbsp; 7.2
?. [Adaptor ligation]
Vortex the master mix and spin down.Aliquot  of the master mix in each well of a 8-PCR tube strip.In the DNA digestion plate (30µL per well), add  of Adapt P1 according to your plate map, from the adaptor plate (see step 1), with a multichannel pipette.Then in all wells, add  of ligation mix and mix by pipetting, seal PCR plate and spin down.Incubate at  .then or at -20°C if not performing the bead purification the day after.
93.5 µl
2.8 µl
7.2 µl
16 °C
4 °C
?. [Bead  purification]
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 ca. 200 bp. This can be achieved by using a volume ratio (bead suspension to sample) of 1.5,  which  is  described  in  the  following  protocol.
?. [Bead  purification]
SeparationPlace the purification plate onto the 96-well magnetic separator.Wait at least   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 (ca 100 µl) by pipetting.Do  not  disturb  the  attracted  beads  while  aspirating  the  supernatant.  Remove the supernatant with the multichannel from the opposite side of the well.Wait at least  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 (ca 100 µl) by pipetting.Do  not  disturb  the  attracted  beads  while  aspirating  the  supernatant.  Remove the supernatant with the multichannel from the opposite side of the well.
?. [Bead  purification]
1st wash with 80 % ethanolPlace 80% (or 70%) ethanol in a 25ml reservoir.With a multichannel pipette, dispense     80% (or 70%)  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.With a multichannel pipette, dispense    80% (or 70%)  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.
200 µl
?. [Bead  purification]
Dry the beadsLet 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.NOTE:  take care not to over dry the bead pellet (bead pellet appears cracked in this case) as this will significantly decrease elution efficiency.
?. [Bead  purification]
Elute DNA fragments libraryTake the purificaation plate from the magnetic stand,  and add  of Tris 10mM pH 8  with a multichannel pipette to resuspend the bead pellet by pipetting up and down 10 times.Incubate the purification plate at room temperature for  .Separate the magnetic beads against the side of the wells by placing the 96-well plate on the  magnetic separator.Wait at least   until all the beads have been attracted by the magnets or until the liquid appears clear.Transfer   of the supernatant containing the purified DNA fragments to a new 96‑well plate. Be careful to avoid pipeting beads during this step. Store the plate at 4°C until and proceed to purification of columns 5-8 and 9-12 similarly (steps 11-15). Seal the plate and store at 4°C  (or store -20°C for a long-term storage) until PCR amplification.Wait at least  until all the beads have been attracted by the magnets or until the liquid appears clear.Transfer  of the supernatant containing the purified DNA fragments to a new 96‑well plate. Be careful to avoid pipeting beads during this step. Store the plate at 4°C until and proceed to purification of columns 5-8 and 9-12 similarly (steps 11-15). Seal the plate and store at 4°C  (or store -20°C for a long-term storage) until PCR amplification.
40 µl
35 µl
?. [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 (see ligation step for details). 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.
?. [PCR amplification to generate Illumina sequencing indexed libraries]
The Reaction mixture for a total volume of 40μL is :  ABCD1&nbsp;conc initialeconc finalvol 1X µl2Q5 buffer NEB5X1x83dNTP mix25mM each0.2 mM0.34primer mix (illPCR1 and illPCR2index)5µM each0.17 µM1.45Q5 hotstart hifi DNA polymerase NEB2u/µl 0.46High GC enhancer NEB5X1X87nuclease-free water  11.98Adaptor-ligated purified template DNA  109total mix  3010TOTAL reaction40
ABCD1&nbsp;conc initialeconc finalvol 1X µl2Q5 buffer NEB5X1x83dNTP mix25mM each0.2 mM0.34primer mix (illPCR1 and illPCR2index)5µM each0.17 µM1.45Q5 hotstart hifi DNA polymerase NEB2u/µl 0.46High GC enhancer NEB5X1X87nuclease-free water  11.98Adaptor-ligated purified template DNA  109total mix  3010TOTAL reaction40
?. [PCR amplification to generate Illumina sequencing indexed libraries]
1st mix preparationDefreeze and vortex all reagents, except enzymes (stored at -20°C), for approximately  Spin down all reagents for approximately  and place on ice.In a 5ml microtube, prepare the 1st mix according to the following table :  ABC1Vol µl 1X µlVol µl for 1 plate (104 reactions)2Q5 buffer 5X88323 dNTP mix 25mM0.331.24Q5 hot start DNA polymerase 2u/µl0.441.65High GC enhancer 5X88326nuclease free water3.3343.27total202080Vortex mix all reagents in the mix and spin down.Aliquot  of the 1st mix into each well of a  8-tube PCR  strip.In a new PCR plate , dispense   of 1st mix with a multichannel in each column.
ABC1Vol µl 1X µlVol µl for 1 plate (104 reactions)2Q5 buffer 5X88323 dNTP mix 25mM0.331.24Q5 hot start DNA polymerase 2u/µl0.441.65High GC enhancer 5X88326nuclease free water3.3343.27total202080
130 µl
20 µl
?. [PCR amplification to generate Illumina sequencing indexed libraries]
2nd mix preparation (primer PCR mix) :prepare 1 mix for each index in a 1.5 ml microtube, (4 mix for a full a plate). for 1 index   ABC1vol 1x µlVol µl for 3 columns2illPCR Primer mix 5µM1.439.23nuclease-free water8.6240.84total10280  Vortex mix all reagents and spin down.In a 12-tubes PCR  strip, aliquot  of the  primer PCR mix into 3 consecutive wells according to the following scheme :In the PCR plate , dispense  of 2nd mix in each line of the plate with a 12 multichannel pipette, onto the 20µl of the first mix.
ABC1vol 1x µlVol µl for 3 columns2illPCR Primer mix 5µM1.439.23nuclease-free water8.6240.84total10280
93 µl
10 µl
?. [PCR amplification to generate Illumina sequencing indexed libraries]
DNA and mix combinationSpin down the ligation purified DNA plate.With a multichannel pipette, transfer   of purified ligated  DNA into the PCR plate and mix by pipetting.Finally, aliquot the  by dispensing  into 3 additional new empty PCR plates.Seal the 4 PCR plates and spin down. The 4 PCR will be performed in parallel In 4 different thermal cyclers, in order to reduce the PCR bias.  ABCD1hot start initial denaturation98°C30s2amplification cycles3denaturation98°C20s4annealing60°C60s15 cycles5extension72°C40s6final extension72°C10min7hold12°CSeal the 4 PCR plates and spin down. The 4 PCR will be performed in parallel In 4 different thermal cyclers, in order to reduce the PCR bias.After PCR, pool back the 4 PCR plates into a single plate with a multichanel.
10 µl
40 µl
10 µl
ABCD1hot start initial denaturation98°C30s2amplification cycles3denaturation98°C20s4annealing60°C60s15 cycles5extension72°C40s6final extension72°C10min7hold12°C
?. [PCR amplification to generate Illumina sequencing indexed libraries]
Final cycle (with pirmers in large excess)Prepare 1 mix for each index in a 1.5 ml microtube  ABCDE1conc stockfinal concentrationvol µl 1Xvol µl for 3 columns2Q5 buffer5X1X0.2243dNTP mix25mM each0.2mM0.089.64illPCR primer mix5µM each0.2µM0.4485nuclease-free water  0.3238.46total  4120Vortex mix all reagents and spin down. In a  12-tube PCR strip, aliquot  of the  mix into 3 consecutive wells according to the following scheme: Dispense of final cycle mix in each line of the plate with a 12 multichannel pipette.Seal the PCR plate and spin down.In a thermocycler, run the final cycle as follows:  ABC1denaturing98°C3 min2annealing60°C2 min3extension72°C12 min4hold12°CPlace at 4°C (or -20°C for a long-term storage).
ABCDE1conc stockfinal concentrationvol µl 1Xvol µl for 3 columns2Q5 buffer5X1X0.2243dNTP mix25mM each0.2mM0.089.64illPCR primer mix5µM each0.2µM0.4485nuclease-free water  0.3238.46total  4120
40 µl
4 µl
ABC1denaturing98°C3 min2annealing60°C2 min3extension72°C12 min4hold12°C
?. [Agarose gel electrophoresis]
Check the efficiency of the PCR on an agarose 1.5% gel .  Load 5µl of each PCR product.
?. [Samples pooling]
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. Proceed to next step.
?. [Size selection with sage science Pippin-Prep]
Perform the size selection of fragments between 300 and 800 pb using a 1,5% DF marker K agarose gel cassette, according to the Pippin prep manufacturer's instructions : Warning :If you perform the size selection on the pool without prior bead clean-up, you may observe a shift between observed and expected size ranges of the smear (approximately 250-750 obtained  instead of 300-800).If you see adaptor dimers on the gel after PCR, it is also preferable to perform a bead clean-up before size selection in the Pippin Prep. In the case you do not have access to a Pippin prep, you can alternatively perform a double size-selection with beads (see the bead manufacturer's instructions for details), or by smear excision and purification from an agarose gel. From our experience, size selection with the Pippin prep is the most accurate and repeatable method, and has to be prefered if you have serveral pools of libraries (for example 3 pools of 192 samples) in order to increase the number of common loci between samples in the ddRAD sub sampling of the genome.
?. [Quality control of the libraries]
Control the quality of the library with a Bioanalyzer (Agilent) 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 :
?. [Quality control of the libraries]
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 BR assay kit, according to the manufacturer's instructions:
?. [Quality control of the libraries]
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.
?. [Quality control of the libraries]
Suggestions to prepare library dilutions : in a 8-tube PCR strip:Prepare intermediate dilutions (1:10 and 1:100) of the library  with the dilution buffer supplied in the qPCR kit.Then prepare the 4 library dilutions to be used in triplicate for qPCR analysis :1:1000 : 10µl of 1/100 +  90µl buffer 1X1:2000 : 50µl of 1/1000 +  50µl buffer 1X1:4000 : 50µl of 1/2000 +  50µl buffer 1X1:8000 : 50µl of 1/4000 +  50µl buffer 1XYou should get more than 10 nM, that is the library concentration usually required by the sequencing platform facilities. The library is now ready for sequencing in single read or paired-end 150 bases in an Illumina sequencer, with one index read. Use the average size of the library size range as estimated from the Bioanalyzer profile to convert DNA concentration from nM  to ng/µl.
?. [Bead  purification]
2nd wash with 80 % ethanol With a multichannel pipette, dispense    80% (or 70%)  ethanol into the purification plate without disturbing the bead pellet. Incubate the purification plate at room temperature for at least 30 s.Carefully remove and discard ethanol by pipetting.
200 µl
?. [Bead  purification]
BindingThis step binds DNA fragments  200bp and larger to the magnetic beads. For the first 4 columns of the digestion-ligation plate, Transfer  of the DNA samples in the first 4 columns of the purification plate containing beads and carefully mix by pipetting up and down 10 times.
40 µl
?. [Bead  purification]
Before starting Prepare    fresh 80% Molecular Biology grade  Ethanol for MN beads (or 70% for AMPure XP beads).Remove the NucleoMag® NGS Bead Suspension (or Beckman Ampure XP) 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 homogeneization and fill all wells of a 8-tube PCR strip with this solution, or use a reagent reservoir.Add   of well-homogeneized bead suspension into the first 4 columns of a new PCR microplate with a multichannel pipette.Spin down the plate containing the digested-ligated products. Fill each well of a 8-PCR tube strip with Tris-Hcl pH8 10mM.Add  of well-homogeneized bead suspension into the first 4 columns of a new PCR microplate with a multichannel pipette.In the DNA digestion plate, add  of Tris 10mM with a multichannel pipette, to be sure that you will be able to take 40µl of digested-ligated DNA.
50 mL
60 µl
5 µl
?. [Samples pooling]
Estimate the double strand DNA concentration in the pool by fluorimetry with a Qubit equipment. If the concentration is too low (less than 20 nM), perform a bead clean-up (ratio 1:1, see above steps 9-16) on a partial volume of the pool to increase the final concentration. Work in a microtube instead of a microplate, with a magnetic microtube stand. Adapt the elution volume to the desired final concentration. Elution should be done in at least 50µl, for subsequent steps.