TITLE: Prokaryotes/Eukaryotes 16S-V4V5 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 16S-V4V5 DNA amplification method applied for both prokaryote and eukaryotes metabarcoding analyses using Illumina Miseq technology. This protocol has been used in many projects studying prokaryotic and eukaryotic diversity (TARA-OCEANS 2009-2013, TARA-PACIFIC 2016-2018), and prokaryote monitoring projects (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 prokaryotic 16SV4V5 primer set 515fY- 926r from Parada et al. 2016.

 
 
 
 

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: 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 16S-V4V5 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
7. PCR Programm :


 

 
In order to reduce the artificial building of chimeras during the PCR process, the # of cycles must be reduced to a minimum : 25, max 30 (chimeras formation mainly 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 Pooled 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 Pooled 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: Pooled 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: Pooled 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: Pooled 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 to your package.
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 16S-V4V5 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
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 pooling
10. Prepare PCR1 Table and pool the triplicate reactions into a single well in a new 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: 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: PCR
6.2. If you work on environmental sample : 
Dispense 24 µL of each tagged-MID-primer PCR mix preparations per well under the PCR hood, and then add 1 µL of the DNA template on the bench.

If you work on strain sample : 
Dispense 21 µL of each tagged-MID-primer PCR mix preparations per well under the PCR hood (so reduce the volume of H2O milliQ in the Master Mix), and then add 4 µL of the DNA template on the bench to get 5-10ng in the MasterMix.
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 (423 pb), no smear.
- It is possible to observe a second band 200 bp longer than the target. This is due to the presence of eukaryotes in the sample.


Store the amplicons at -20°C until PCR purification.
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. PCR Mix preparation :
SECTION: PCR1 protocol
6.1. Prepare the Master Mix :




 

SECTION: PCR1 protocol
6.2. If you work on environmental sample : 
Dispense 24 µL of each tagged-MID-primer PCR mix preparations per well under the PCR hood, and then add 1 µL of the DNA template on the bench.

If you work on strain sample : 
Dispense 21 µL of each tagged-MID-primer PCR mix preparations per well under the PCR hood (so reduce the volume of H2O milliQ in the Master Mix), and then add 4 µL of the DNA template on the bench to get 5-10ng in the MasterMix.
SECTION: PCR1 protocol
7. PCR Programm :


 

 
 
In order to reduce the artificial building of chimeras during the PCR process, the # of cycles must be reduced to a minimum : 25, 30, max 32 (chimeras formation mainly 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: 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 (467 bp), no smear, for prokaryotes.
- It is possible to observe a second band 200 bp longer than the target. This is due to the presence of eukaryotes in the sample. 


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: PCR
5. PCR plate's plan :

Each DNA sample (DNA1, DNA2, DNA3...) will be amplified with its own tagged-MID-primer (515fY-M8R001, 515fY-M8R004, 515fY-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).