TITLE: Eukaryotes 18S-V4 rRNA Metabarcoding PCR protocol for NGS Illumina sequencing 
AUTHORS: Sarah  Romac

[DESCRIPTION]
Nowadays metabarcoding approaches allow to explore the diversity of different communities using next-generation sequencing (NGS).

Here we describe the 18S-V4 DNA amplification method applied for eukaryote metabarcoding analyses using Illumina Miseq technology. This protocol has been used in many projects studying eukaryotic diversity (TARA-OCEANS 2009-2013, TARA-PACIFIC 2016-2018), and eukaryote monitoring projects (MOOSE-GE 2017-..., ROSCOFF ASTAN 2009-ongoing).
 

We developed the flowchart for 2 different sequencing platforms : Fasteris-Gene Support SA (Plan-Les-Ouates, Swiss) and GeT-PlaGE (Toulouse, France).

[BEFORE_START]
Do aliquots of 1 mL of sterile milliQ water.

Before starting, place all the supplies and sterile milliQ Water needed for the PCR under the PCR hood and switch on the UV light for at least 20 min.

[GUIDELINES]
As metabarcoding is very sensitive to contaminations by exogen DNA, please respect some conditions :
- always wear a labcoat, and clean nitrile gloves;
- separate the work area for prePCR and postPCR manipulations.
- do your PCR under a PCR hood.

[STEPS]
SECTION: Tagged Primer Design and preparation
1. We use the eukaryotic 18SV4 primer set TAReukF1- TAReukR3 from Stoeck et al. 2010.

  
  

SECTION: PCR
4. PCR reactions are performed using the Taq polymerase Phusion High-Fidelity Master Mix with GC buffer (Thermofisher, Cat No F-532 L).
This Taq  has a good proof-reading and its buffer allows amplification of high GC templates.

Keep the same annealing temperature as the one is used with the usual 18S-V4 primer set.

First test each of your taggeg-primer sets on a positive and a negative control following steps 5, 6, 7 and 8.

Then you can perform DNA sample amplification following next steps.
SECTION: PCR
6. PCR Mix preparation :

SECTION: PCR
5. PCR plate's plan :

Each DNA sample (DNA1, DNA2, DNA3...) will be amplified with its own tagged-MID-primer (V4f-M8R001, V4f-M8R004, V4f-M8R005...). So there will be as many PCR mix preparations as there are DNA samples to amplify.

In order to get enough material (50 ng), triplicate the PCR reactions on each DNA sample (you will pool them after).

One positive control and one negative control will be added for each PCR mix preparation.

PCR reactions are prepared on a semi-skirted 96-wells PCR plate (like AB-0900 PCR plate).


  

SECTION: PCR
7. PCR Programm :

  
In order to reduce the artificial building of chimeras during the PCR process, you should reduce the # of cycles to a minimum : 20, 25, max 30 (chimerization occurs principally during the plateau-phase of the PCR reaction).

SECTION: PCR
8. Check the quality of all the PCR products on an 1.2 % agarose gel :

  

 


SECTION: PCR product purification
9. PCR products are purified using the purification kit: NucleoSpin® PCR Clean-Up (Macherey-Nagel, cat. nb 740609.50 or 740609.250 ).

Store the kit at room temperature.

  
SECTION: PCR product purification
10. Prepare Purification Run Table and pool the triplicate PCR into a single microtube with appropriate labelling (sample, target and tag-MID nb, PCR date).

  

SECTION: PCR product purification
11. Mix 1 vol of sample with 2 volumes of buffer NT.
Follow the instructions of manufacturer (mentioned in the Step 9), except for the elution step.

Elution Step : 

- Place the NucleoSpin PCR Clean-Up Column into a clean 1.5mL microtube correctly labeled (sample, target tagged-MID, date).
- Add 22 µL buffer NE preheated at 65°C directly onto the column and incubate 5 min @ 65°C.
- Centrifuge 1 min @ 11 000 g.

Store the purified PCR products at -20°C or directly do the quantification.
SECTION: PCR products quantification
12. PCR products are quantified using the quantification kit: Quant-iT™ PicoGreen® dsDNA reagent *2000 assays* (Invitrogen, cat nb P7581) and a Fluorometer Plate readerFluorometer Plate reader, following the manufacturer's protocol.


  

SECTION: Final Pool Amplicons preparation
13. After quantification of the PCR products, amplicons will be pooled before shipment to  the NGS Fasteris sequencing platform.

One final tube (called "Pooled Amplicons") will contain all pooled amplicons at equimolar concentration, ready for the library preparation. The amounts and volume required by Fasteris are : 1μg of equimolar amplicon pool in 30 μl (so Pool Amplicons concentration should be >35 ng/µL) .

The volume of each amplicon that will added in the tube "Pooled Amplicons" is calculated based on their average concentration as follows :

Calculation table for Pool Amplicons preparation :

  

If the final volume of Pooled Amplicons is higher than 30 µL, (so concentration inferior to 35ng/µL), an additionnal concentration step will be necessary. For this we used the purification kit: NucleoSpin® PCR Clean-Up (Macherey-Nagel, cat. nb 740609.50 or 740609.250 ).


SECTION: Pool Amplicons Quantification and Quality Checking
14. After the preparation of the Pooled Amplicons (and concentration if needed), the final concentration is checked by quantification using Qubit 4 Fluorometer (Invitrogen) with the kit Qubit 1x dsDNA HS Assay (Invitrogen, Thermofisher Scientific cat. No Q33230).
  

SECTION: Pool Amplicons Quantification and Quality Checking
15. If possible, check the final Pooled Amplicons quality on a Bioanalyzer using the kit Agilent DNA 1000 (Agilent Technologies, Cat. No 5067-1504).

  

SECTION: Pool Amplicons Quantification and Quality Checking
16. Store the Pooled Amplicons at -80°C until the shipment to Fasteris.
SECTION: Shipment Conditions to Fasteris
17. Pooled Amplicons must be shipped via Dry Ice to the following address :
FASTERIS SA
NGS Services
Chemin du Pont-du-Centenaire 109
CH-1228 Plan-les-Ouates
Switzerland

Additonnal documents to include in the package (that must also be sent by e-mail to Fasteris NGS services (ngs@fasteris.com) :
- Quotation number Q##### ;
- Order form ;
- Purchase oder edited by your company.

Don't forget to add a Pro Forma Invoice in the package.

SECTION: Tagged Primer Design and preparation
None. We purchase our Tagged-primers at Eurogentec, using the RP-Cartridge Gold purification. Primers are delivered lyophilised.

Dried primers are eluted at 100 µM with TE 1X sterile buffer under a PCR hood.
We let primer dissolution for 15 min at room temperature. Short vortex and spin.
Primer working solutions are then prepared at 10 µM with sterile milliQ water molecular grade.

Stock primers and primer working solutions are stored at -20°C.
SECTION: PCR1 protocol
None. PCR reactions are performed using the Taq polymerase Phusion High-Fidelity Master Mix with GC buffer. This Taq  has a good proof-reading and its buffer allows amplification of high GC templates.

First test each of your taggeg-primer sets on a positive and a negative control.

Keep the same annealing temperature as the one is used with the usual 18S-V4 primer set.
SECTION: PCR1 protocol
None. PCR Mix preparation :



  
Dispence 24 µL of PCR mix preparation per well under the PCR hood, and then add 1 µL of template on the bench.

In order to get enough material (50 ng), triplicate the PCR reactions on each DNA sample (you will pool them after).

SECTION: PCR1 protocol
None. PCR plate's plan :

   

SECTION: PCR1 protocol
None. PCR Programm :

  
In order to reduce the artificial building of chimeras during the PCR process, you should reduce the # of cycles to a minimum : 20, 25, max 30 (chimerization occurs principally during the plateau-phase of the PCR reaction).

SECTION: PCR1 protocol
None. Check the quality of all your PCR products on an 1.2 % agarose gel.

Amplifications worked very well if :
- Negative control has not amplified;
- Positive control amplified;
- Sample amplifications have single band at the good size (471 pb), no smear.

Store your amplicons at -20°C until PCR purification.

Minimum conditions required by Genotoul platform :
V total (PCR1) > 50µL
C (PCR1) > 20ng/µL (you can approximatively estemiated with the intensity of the band on the agarose gel)


SECTION: PCR1 pooling
None. Prepare PCR1 Table and pool the triplicate reactions into a single well in a semi-skirted PCR plate with appropriate labelling (Run ID, date).

   



Seal the Plate containing the amplicons pools to avoid evaporation risk and store it at -20°C until shipment.
SECTION: PCR1 pooling
None. Plate containing the pooled amplicons must to be shipped via Dry Ice to the following address :
Plateforme GeT-Biopuces
INSA/TBI
Bât 39 - Bio5
135 Avenue de Rangueil
31077 Toulouse Cedex 04
FRANCE

Additonnal documents to include in the package (and also send them by mail) :
- sample file PCR1 (= plate's plan, and amplicon's size);
- Miseq prestation file completed and signed
NB : These files are given by the platform.
SECTION: Tagged Primer Design and preparation
3. Lyophilized Tagged-primers are obtained at Eurogentec, using the RP-Cartridge Gold purification.

Work always under the PCR hood.
SECTION: Tagged Primer Design and preparation
3.1. Elute dried primers at 100 µM with TE 1X sterile buffer under a PCR hood.
SECTION: Tagged Primer Design and preparation
3.2. Primer dissolution is done for 15 min at room temperature under the hood.
Short vortex and spin.
SECTION: Tagged Primer Design and preparation
3.3. Primer working solutions are then prepared at 10 µM with sterile milliQ water molecular grade.

For each MID-primer, add 10 µL of 100 µM of Stock primer to 90 µL in a 1.5mL sterile microtube correctly labeled (MID###, concentration, date, operator).
SECTION: Tagged Primer Design and preparation
3.4. Stock primers and primer working solutions are stored at -20°C.
SECTION: Tagged Primer Design and preparation
2. 
SECTION: Tagged Primer Design and preparation
3. Lyophilized Tagged-primers are obtained at Eurogentec, using the RP-Cartridge Gold purification.

Work always under the PCR hood.
SECTION: PCR1 protocol
4. PCR reactions are performed using the Taq polymerase Phusion High-Fidelity Master Mix with GC buffer (Thermofisher, Cat No F-532 L).
This Taq  has a good proof-reading and its buffer allows amplification of high GC templates.

Keep the same annealing temperature as the one is used with the usual 18S-V4 primer set.

First test each of your taggeg-primer sets on a positive and a negative control following steps 5, 6, 7 and 8.

Then you can perform DNA sample amplification following next steps.
SECTION: PCR1 protocol
6. PCR Mix preparation :

SECTION: PCR1 protocol
5. PCR plate's plan :

In order to get enough material (50 ng), triplicate the PCR reactions on each DNA sample (you will pool them after).

One positive control and one negative control will be added for each PCR plate.

PCR reactions are prepared on a semi-skirted 96-wells PCR plate (like AB-0900 PCR plate).

   

SECTION: PCR1 protocol
7. PCR Programm :

  
In order to reduce the artificial building of chimeras during the PCR process, you should reduce the # of cycles to a minimum : 25, max 30 (chimerization occurs principally during the plateau-phase of the PCR reaction).

SECTION: PCR1 protocol
8. Check the quality of all the PCR products on an 1.2 % agarose gel.


  


 

SECTION: Tagged Primer Design and preparation
None. Tagged-Primer Design for GeT-PLaGE platformFor amplicons sequenced by Genotoul platform, primers are built with 1/2sequence with the Illumina Miseq 2*250pb adaptors. 



 



SECTION: Tagged Primer Design and preparation
None. 
SECTION: Tagged Primer Design and preparation
3.1. Elute dried primers at 100 µM with TE 1X sterile buffer under a PCR hood.
SECTION: Tagged Primer Design and preparation
3.2. Primer dissolution is done for 15 min at room temperature under the hood.
Short vortex and spin.
SECTION: Tagged Primer Design and preparation
3.3. Primer working solutions are then prepared at 10 µM with sterile milliQ water molecular grade.

For each MID-primer, add 10 µL of 100 µM of Stock primer to 90 µL in a 1.5mL sterile microtube correctly labeled (MID###, concentration, date, operator).
SECTION: Tagged Primer Design and preparation
3.4. Stock primers and primer working solutions are stored at -20°C.
SECTION: PCR1 protocol
6.1. Prepare the Master Mix :
 

  

SECTION: PCR1 protocol
6.2. Dispence 24 µL of PCR mix preparation per well under the PCR hood, and then add 1 µL of template on the bench.

SECTION: PCR1 protocol
8.1. Prepare 1,2 % agarose  gel in TAE 0.5x buffer  :

- In a Becher, put 1.2g agarose (Interchim, ref 31272L ) in 100mL TAE 0.5x buffer (TAE prediluted in milliQ water from TAE 10x-Thermofisher Scientific, ref 15558042).

- Heat under total dissolution of the agarose powder (you can use a microwave or a stirer plate).

- Add one drop of Ethidium Bromide (Eurobio, ref GEPBET02AF).

- Prepare casting tray with combs according to your number of PCR products to check. (Biorad, ref 1704484)

  

- Poor gel in the casting tray and check there are no bubbles.

- Let solidify for 20 min.
SECTION: PCR1 protocol
8.2. Sample loading and electrophoresis conditions :

Prepare  the loading samples :
In a semi-skirted 96-wells PCR plate (like AB-0900 PCR plate), mix 5µL of each sample with 1µL of loading buffer 6x (Thermofisher Scientific, ref R0611). 

Place solidified gel in the proper orientation (electrophoresis occurs from cathode to anode).

Load :
- PCR products : 6µL ;
- Smartladder 200 to 10 000 bp : 3µL ; (Eurogentec, ref MW-1700-10).

Close the caster and connect it to the generator ( Bio-Rad, ref 1645050).

Let run the electrophoresis at 110 V for 45 min.
SECTION: PCR1 protocol
8.3. Amplification result observation :

After migration, observe amplification results under UV light using an Imager (for instance : ImageQuant LAZ4000, GE Healthcare).
  

Amplifications worked very well if :
- Negative control has not amplified;
- Positive control has amplified;
- Amplifications have band at the good size (471 pb), no smear.


Store the amplicons at -20°C until PCR purification.
SECTION: PCR1 pooling
9. Minimum conditions required by Genotoul platform :

Total volume  (PCR1) > 50µL
Concentration (PCR1) > 20ng/µL (can be roughly estimated by the intensity of the band on the agarose gel)
SECTION: PCR1 pooling
10. Prepare PCR1 Table and pool the triplicate reactions into a single well in a semi-skirted PCR plate with appropriate labelling (Run ID, date).

 
Seal the Plate containing the amplicons pools to avoid evaporation risk and store it at -20°C until shipment.  
SECTION: PCR1 pooling
11. Plate containing the pooled amplicons must to be shipped via Dry Ice to the following address :
Plateforme GeT-Biopuces
INSA/TBI
Bât 39 - Bio5
135 Avenue de Rangueil
31077 Toulouse Cedex 04
FRANCE

Additonnal documents to include in the package (that must also be sent by e-mail) :
- sample file PCR1 (= plate's plan, and amplicon's size);
- Miseq prestation file completed and signed
NB : These files are given by the platform.
SECTION: PCR
6.1. Prepare the Master Mix :


  

SECTION: PCR
6.2. Dispence 24 µL of each tagged-MID-primer - PCR mix preparation per well under the PCR hood, and then add 1 (5ng) µL of template on the bench.
SECTION: PCR
8.1. Prepare 1,2 % agarose  gel in TAE 0.5x buffer  :

- In a Becher, put 1.2g agarose (Interchim, ref 31272L ) in 100mL TAE 0.5x buffer (TAE prediluted in milliQ water from TAE 10x-Thermofisher Scientific, ref 15558042).

- Heat under total dissolution of the agarose powder (you can use a microwave or a stirer plate).

- Add one drop of Ethidium Bromide (Eurobio, ref GEPBET02AF).

- Prepare casting tray with combs according to your number of PCR products to check. (Biorad, ref 1704484)

  

- Poor gel in the casting tray and check there are no bubbles.

- Let solidify for 20 min.
SECTION: PCR
8.2. Sample loading and electrophoresis conditions :

Prepare  the loading samples :
In a semi-skirted 96-wells PCR plate (like AB-0900 PCR plate), mix 5µL of each sample with 1µL of loading buffer 6x (Thermofisher Scientific, ref R0611). 

Place solidified gel in the proper orientation (electrophoresis occurs from cathode to anode).

Load :
- PCR products : 6µL ;
- Smartladder 200 to 10 000 bp : 3µL ; (Eurogentec, ref MW-1700-10).

Close the caster and connect it to the generator ( Bio-Rad, ref 1645050).

Let run the electrophoresis at 110 V for 45 min.
SECTION: PCR
8.3. Amplification result observation :

After migration, observe amplification results under UV light using an Imager (for instance : ImageQuant LAZ4000, GE Healthcare).
  

Amplifications worked very well if :
- Negative control has not amplified;
- Positive control has amplified;
- Amplifications have band at the good size (427 pb), no smear.


Store the amplicons at -20°C until PCR purification.