Construction of individuals ddRADseq libraries for macro-algae (Kelp) V.3

5 M sodium chloride solution (NaCl)

29.2g

Dissolve the slat in MilliQ water and fill up to 100mL.

Autoclave.

Store at Room temperature

Annealing buffer stock (10x)

Annealing buffer composed 100 mM Tris-HCl, pH8; 500 mM NaCl and 10 mM EDTA

5mL

5mL

1mL

39mL

Homogenize and autoclave.

Store at Room temperature

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) :

Barcoded_P1_adaptors.xlsx

In a PCR plate wells, combine each oligo P1_PstI_x.F with its complementary oligo P1_PstI_x.R :

4µL

4µL

10µL

82µL

A	B	C	D	E	F	G	H
P1_PstI_01			P1_PstI_09			P1_PstI_17
P1_PstI_02			P1_PstI_10			P1_PstI_18
P1_PstI_03			P1_PstI_11			P1_PstI_19
P1_PstI_04			P1_PstI_12			P1_PstI_20
P1_PstI_05			P1_PstI_13			P1_PstI_21
P1_PstI_06			P1_PstI_14			P1_PstI_22
P1_PstI_07			P1_PstI_15			P1_PstI_23
P1_PstI_08			P1_PstI_16			P1_PstI_24

Example of a plate map for barcoded P1 adaptors. Allow enough space betweeen the rows to avoid cross-contaminations between barcodes.

The reaction is performed in a thermocycler with the following PCR cycling conditions :

A	B	C	D
Cycle step	Temperature	Time	Cycles
Initial Denaturation	97.5°C	2.5 min	1
Annealing	96°C (-3°C per cycle)	1 min	25
Hold	4°C

PCR cycling conditions

Store at4°C (or at-20°C for a long-term storage)

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) :

No-Barcoded_P2_adaptors.xlsx

In 1.5mL microtube, combine oligo P2_HhaI.F with its complementary oligo P2_HhaI.F (or P2_MseI.F and P2_MseI.R)

400µL

400µL

100µL

100µL

Then aliquot this volume into 125µL in each well of a 8- PCR tube strip.

The reaction is performed in a thermocycler with the following PCR cycling conditions :

A	B	C	D
Cycle step	Temperature	Time	Cycles
Initial Denaturation	97.5°C	2.5 min	1
Annealing	96°C (-3°C per cycle)	1 min	25
Hold	4°C

PCR cycling conditions

Pool all reaction in a1.5mL tube.

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

In 24 1.5mL 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 .

5µL ILLPCR1 oligo forward (100µM)

5µL ILLPCR2 oligo reverse (100µM) ind01 to ind27 (one per tube)

90µL 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) :

Illumina_indexed_primers.xlsx

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).

In a Rack of Tube Strips (consumable of NucleoSpin 96 Plant II kit) add15mg to 20mg of each sample with one 3 mm grinding ball stainless steel. Close the Tubes Strips with Cap Strips.

Grind dry tissues using Mixer Mill MM400 Retch using 2 cycles of 0h 2m 0s at maximum frequency.

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.

Instruction-NucleoSpin-96-Plant-II.pdf

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

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.

Transferring the 120µL into 1.5 mL microtubes. Add nuclease free water to fill up to 400µL.

The purifications were performed according to the manufacturer's instructions with elution into 30 μL (2 x 15 µL) of the supplied elution buffer.

Instruction-NucleoSpin-gDNA-Clean-up-XS.pdf

Removal of residual ethanol and concentration were performed by incubation 0h 15m 0s at 70°C

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)

Quality Control of Genomic DNA

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

Quantification of Genomic DNA ( Preparation for one 96 wells PCR plate) using PicoGreen‱ ^TM

Quantify Genomic DNA extract using PicoGreen^TM.

Qant-iT™ PicoGreen® dsDNA Quantification

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

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

Equipment

Value	Label
Synergy 2	NAME
absorbance microplate reader	TYPE
BioTek	BRAND
Synergy2	SKU

Equipment

Value	Label
SPARK	NAME
Microwell plate reader	TYPE
TECAN	BRAND
SPARK	SKU

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.

Preparation of of 1X TE buffer 11mL of 1X TE buffer

In 15mL

550µL

10.450mL

Mix by inverting the tube several times.

Preparation of DNA solution at 5000 pg/µl (for 3 ranges)

In 0.5mL

4µL

76µL

Mix by inverting the tube several times.

Preparation of the standard range 0 pg/µl to 1000 pg/µl

Prepare the following standard mixture in 8 0.5mL

A	B	C	D	E
Tubes	Standard DNA solution concentration (pg/µL)	Standard DNA solution volume (µL)	1X TE (µL)	Final DNA concentration (pg/µL)
1	5000	42	168	1000
2	5000	21	189	500
3	5000	9	171	250
4	1000	21	189	100
5	500	21	189	50
6	100	18	162	10
7	50	18	162	5
8	0	0	180	0

Standard DNA solutions preparation Pipette 50µL of each standard mixture in the first two columns of the black, sterile, 96-well plate :

A	B	C	D	E	F	G	H
1000 pg/µL	1000 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA
500 pg/µL	500 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA
250 pg/µL	250 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA
100 pg/µL	100 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA
50 pg/µL	50 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA
10 pg/µL	10 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA
5 pg/µL	5 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA
0 pg/µL	0 pg/µL	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA	unknown DNA

Exemple of map plate for PicoGreen‱ quantification

Pipette 49 µl of 1X TE buffer in the remaining wells.

49µL

Pipette 1 µl of the unknown DNA samples in the remaining wells.

1µL

Prepare PicoGreen‱ work solution ^® work solution

In 10mL

25µL

4.975mL

Mix and protect from light.

Pipette 50µL in each well, including the standard and unknown sample wells.

Protect the 96-well plate from light and incubate for 0h 5m 0s at room temperature.

In a PCR plate, put around 100ngof genomic DNA in a volume of 40µL (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.

Double digest around 100ng of high quality genomic DNA with selected restriction enzymes 50µLreaction 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.

Vortex all reagents, except enzymes (stored at -20°C), for approximately 0h 0m 5s

Spin down all reagents for approximately 0h 0m 5s and placeOn ice.

In a microtube, prepare the digestion mix, according to the following table for a total volume of 50µL:

A	B	C	D	E
	Initial concentration	Final concentration	n=1	n=100 (1 plate)
Genomic DNA		~100 ng	40 µL
Cutsmart buffer	10X	1X	5 µL	500 µL
Enzyme 1 (PstI HF)	20 u/µL	10U	0.5 µL	50 µL
Enzyme 2 (HhaI or MseI)	20 u/µL	10U	0.5 µL	50 µL
nuclease-free water			4 µL	400 µL
TOTAL			50 µL	1000 µL

Digestion master mix composition

Vortex the master mix and spin down.

Aliquot 125µL of the digestion master mix in each well of a 8-PCR tube strip.

In the DNA plate (containing 40µL per well), add10µL of digestion master mix with a x8 multichannel pipette and mix by pipetting, seal PCR plate and spin down.

Incubate at 37°C 0h 0m 5s

Then store at 4°C

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.

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.

Before starting

Prepare 50mL of fresh 80% Molecular Biology Grade Ethanol

40mL

10mL

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.

Binding

This step binds DNA fragments 100 bp and larger to the magnetic beads.

Pipette 45µL of NGS Beads suspension with x8 multichannel pipette and transfer in digestion plate (plate with 45µL of digested template DNA for each sample), carefully mix by pipetting up and down 10 times.

Incubate 0h 5m 0s atRoom temperature

Separation

Place the purification plate onto the 96-well magnetic separator.

Wait at least 0h 5m 0s 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.

1st wash with 80 % ethanol

Place 80% ethanol in a reagent reservoir.

With a x8 multichannel pipette, dispense200µL of 80% ethanol into the purification plate without disturbing the bead pellet.

Incubate the purification plate at room temperature for at least 0h 0m 30s

Carefully and completely remove and discard ethanol by pipetting.

2nd wash with 80 % ethanol

With a x8 multichannel pipette, dispense200µL of 80% ethanol into the purification plate without disturbing the bead pellet.

Incubate the purification plate at room temperature for at least 0h 0m 30s

Carefully and completely remove and discard ethanol by pipetting.

Dry the beads

Let the purification plate on the magnetic separator and incubate at Room temperature for maximum 0h 5m 0s in order to allow the remaining traces of ethanol to evaporate.

Elute DNA fragments

Take the purification plate from the magnetic stand, and add 40µL 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 0h 5m 0s .

Separate the magnetic beads against the side of the wells by placing the 96-well plate on the magnetic separator.

Wait at least 0h 5m 0s until all the beads have been attracted by the magnets or until the liquid appears clear.

Transfer 35µL 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.

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

Vortex all reagents, except enzymes (stored at -20°C), for approximately 0h 0m 5s

Spin down all reagents for approximately 0h 0m 5s and placeOn ice.

In a microtube, prepare the ligation mix, according to the following table for a total volume of 60µL:

A	B	C	D	E
	Initial concentration	Final concentration	n=1	n=100 (1 plate)
Digested purified template DNA + P1 adaptor			40 µL
P2 adaptor (HhaI or MseI)	40 µM	330 nM	0.5 µL	50 µL
T4 ligase buffer	10X	1X	6 µL	600 µL
T4 ligase	400 u/µL	160U	0.4 µL	40 µL
nuclease-free water			13.1 µL	1310 µL
TOTAL			60 µL	2000 µL

Ligation master mix composition

Vortex the master mix and spin down.

Aliquot 125µL of the ligation master mix in each well of two 8-PCR tube strip.

In the digested purified plate (containing 35µL of digested purified template DNA and 5µLof barcoded P1 adaptors ), add20µL of ligation master mix with a x8 multichannel pipette and mix by pipetting, seal PCR plate and spin down.

Incubate at 16°C 0h 0m 5s

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

Before starting

Prepare 50mL of fresh 80% Molecular Biology Grade Ethanol

40mL

10mL

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.

Binding

This step binds DNA fragments 100 bp and larger to the magnetic beads.

Pipette 60µL of NGS Beads suspension with x8 multichannel pipette and transfer in adaptor-ligated plate (plate with 60µL of digested and adaptor-ligated template DNA for each sample), carefully mix by pipetting up and down 10 times.

Incubate 0h 5m 0s atRoom temperature

Separation

Place the purification plate onto the 96-well magnetic separator.

Wait at least 0h 5m 0s 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.

1st wash with 80 % ethanol

Place 80% ethanol in a reagent reservoir.

With a x8 multichannel pipette, dispense200µL of 80% ethanol into the purification plate without disturbing the bead pellet.

Incubate the purification plate at room temperature for at least 0h 0m 30s

Carefully and completely remove and discard ethanol by pipetting.

2nd wash with 80 % ethanol

With a x8 multichannel pipette, dispense200µL of 80% ethanol into the purification plate without disturbing the bead pellet.

Incubate the purification plate at room temperature for at least 0h 0m 30s

Carefully and completely remove and discard ethanol by pipetting.

Dry the beads

Let the purification plate on the magnetic separator and incubate at Room temperature for maximum 0h 5m 0s in order to allow the remaining traces of ethanol to evaporate.

Elute DNA fragments

Take the purification plate from the magnetic stand, and add 40µL 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 0h 5m 0s .

Separate the magnetic beads against the side of the wells by placing the 96-well plate on the magnetic separator.

Wait at least 0h 5m 0s until all the beads have been attracted by the magnets or until the liquid appears clear.

Transfer 35µL 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.

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 40µL is :

A	B	C	D
	Initial concentration	Final concentration	n=1
Adaptor-ligated purified template DNA			10 µL
Primer mix (ILLPCR1 and ILLPCR2ind)	5 µM each	0.17 µM	1.36 µL
Q5 buffer	5X	1X	8 µL
High GC enhancer	5X	1X	8 µL
dNTP mix	25mM each	0.20 µM	0.32 µL
Q5 hotstart hifi polymerase	2 u/µL	0.8 U	0.40 µL
nuclease-free water			11.92 µL
Total mix			30 µL
TOTAL reaction			40 µL

PCR mixture composition

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

First PCR mix preparation (with primers in limiting quantity)

Defreeze and vortex all reagents, except enzymes (stored at -20°C), for approximately0h 0m 5s

Spin down all reagents for approximately0h 0m 5s and place On ice.

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

A	B	C	D	E
	Initial concentration	Final concentration	n=1	n=10 (one column of 1 plate)
Adaptor-ligated purified template DNA			10 µL
Primer mix (ILLPCR1 and ILLPCR2ind)	5 µM each	0.17 µM	1.36 µL	13.6 µL
Q5 buffer	5X	1X	8 µL	80 µL
High GC enhancer	5X	1X	8 µL	80 µL
dNTP mix	25mM each	0.20 µM	0.32 µL	3.2 µL
Q5 hotstart hifi polymerase	2 u/µL	0.8 U	0.40 µL	4 µL
nuclease-free water			11.92 µL	119.2 µL
TOTAL			30 µL	300 µL

PCR mix composition

Vortex mix all reagents in the mix and spin down.

In a new PCR plate, dispense 30µL of 1st mix in each column.

DNA and mix combination

Spin down the adaptor-ligated purified template DNA plate.

With a multichannel pipette, transfer 10µL of adaptor-ligated purified template DNA into the PCR plate and mix by pipetting.

Finally, aliquot the 40µL of the total mix by dispensing 20µL 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.

A	B	C	D
Cycle step	Temperature	Time	Cycles
Hot start initial denaturation	98°C	30 sec	1
Denaturation	98°C	20 sec	15
Annealing	60°C	30 sec	15
Extension	72°C	40 sec	15
Final extension	72°C	10 min	1
Hold	4°C

PCR program for the Illumina indexing PCR

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

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) :

A	B	C	D	E
	Initial concentration	Final concentration	n=1	n=10 (one colomn of 1 plate)
Primer mix (ILLPCR1 and ILLPCR2ind)	5 µM each	3.35 µM	2.68 µL	26.8 µL
Q5 buffer	5X	1X	0.80 µL	8 µL
dNTP mix	25mM each	0.20 µM	0.32 µL	3.2 µL
nuclease-free water			0.20 µL	2 µL
TOTAL			4 µL	40 µL

Final cycle PCR mix composition

Mix all reagents by pipetting and spin down.

Dispense 4µL 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 :

A	B	C	D
Cycle step	Temperature	Time	Cycles
Denaturing	98°C	3 min	1
Annealing	60°C	2 min	1
Extension	72°C	12 min	1
Hold	12°C

PCR program for the final cycle of the illumina PCR

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

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

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 5µL of all individuals in a single low binding 1.5mL 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 1.5mLmicrotubes .

Vortex mix and spin down.

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

Before starting

Prepare 10mLof fresh 80% Molecular Biology Grade Ethanol

8mL

2mL

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.

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 0h 5m 0s atRoom temperature

Separation

Place the purification tube(s) onto the magnetic microtube stand.

Wait at least 0h 5m 0s 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.

1st wash with 80 % ethanol

Dispense1mL of 80% ethanol into the purification tube(s) without disturbing the bead pellet.

Incubate the purification tube(s) at room temperature for at least 0h 0m 30s

Carefully and completely remove and discard ethanol by pipetting.

2nd wash with 80 % ethanol

Dispense1mL of 80% ethanol into the purification tube(s) without disturbing the bead pellet.

Incubate the purification tube(s) at room temperature for at least 0h 0m 30s

Carefully and completely remove and discard ethanol by pipetting.

Dry the beads

Let the purification tube(s) on the magnetic separator and incubate at Room temperature for maximum 0h 5m 0s in order to allow the remaining traces of ethanol to evaporate.

Elute DNA fragments

Take the purification tube(s) from the magnetic stand, and add 100µL 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 0h 5m 0s .

Separate the magnetic beads against the side of the tube by placing the tube(s) on the magnetic separator.

Wait at least 0h 5m 0s until all the beads have been attracted by the magnets or until the liquid appears clear.

Transfer 90µL of the supernatant in a new(s) low binding 1.5mL 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.

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.

Qubit_dsDNA_HS_Assay_UG.pdf

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

Vortex mix and spin down.

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

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 : 

Pippin-prep-Quick-Guide-CDF1510-marker-K3.pdf

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 :

Agilent_high_SensitivityDNA_KG_EN.pdf

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:

Qubit_dsDNA_HS_Assay_UG.pdf

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).

NEBNext_manualE7630.pdf

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.

Suggestions to prepare library dilutions for qPCR

In 0.5mL 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 : 1µL of library + 99µL of 1X buffer

1:1 000 : 10µL of library + 90µL of 1X buffer

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

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

1:30 000 : 50µL of 1:10 000 dilution + 100µL 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 :

nM_ngµL_Conversion_Calculator.xlsx

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.