Sep 20, 2024
Version 2
Prokaryotes/Eukaryotes 16S-V4V5 rRNA Metabarcoding PCR protocol for NGS Illumina sequencing V.2
- 1UMR7144-Roscoff Marine Station
- Ecology of Marine Plankton (ECOMAP) team - Roscoff
- Protist Research to Optimize Tools in Genetics (PROT-G)
Protocol Citation: Sarah Romac 2024. Prokaryotes/Eukaryotes 16S-V4V5 rRNA Metabarcoding PCR protocol for NGS Illumina sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq42o3vk5/v2Version created by Sarah Romac
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 09, 2021
Last Modified: September 20, 2024
Protocol Integer ID: 108094
Keywords: PCR, NGS, metabarcoding, prokaryotes, 16S-V4V5
Abstract
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).
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.
Materials
Specific Equipment (more details in the concerned steps) :
- PCR hood equipped with UV light and HEPA filter ;
- Thermocycler.
- Qubit 4 Fluorometer (Invitrogen) ;
- Fluorometer Plate reader ;
- Gel Tray Caster and Imager .
Optional Equipment :
- 2100 Bioanalyzer Instrument (Agilent)
Supplies :
Sterile microtubes 1,5mL
Semi-skirted PCR plates 0.2mL, 96 wells (like AB-0900) and thermoresistant seals.
Filter tips
Reagents and kis are mentioned in the protocol in the concerned steps.
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.
Tagged Primer Design and preparation
Tagged Primer Design and preparation
We use the prokaryotic 16SV4V5 primer set 515fY- 926r from Parada et al. 2016.
Location of the V4V5 part on the 16S structure.
Tagged-Primer Design for Fasteris platform24 steps
Amplicons from each DNA sample are all pooled in a single microtube. Each pool of amplicons will be considered as a "library sample " or called "Pooled Amplicons" and loaded on a Miseq 2x250 run.
To allow the latter separation of each sample in the Pooled Amplicon sample, each DNA is amplified using forward tagged primer built with a structure 5'-NNNN-MID-forwardprimer'. The reverse primer is not modified.
The tag, or Multiplex IDentifier (MID) is a unique short sequence of 7 or 8 bases compatible with the forward primer V4f and generated using the matrix oligoTag program (Coissac et al. 2012). We also added 4 N at the 5' extremity of the forward primer to help MID sequence conservation during the cluster synthesis step on the Flowcell.
Figure 2 : Fasteris tagged-primer description.
Figure 3 : MID list adapted for 16S-V4V5 primer set.
Lyophilized Tagged-primers are obtained at Eurogentec, using the RP-Cartridge Gold purification.
Work always under the PCR hood.
Elute dried primers at 100 µM with TE 1X sterile buffer under a PCR hood.
Primer dissolution is done for 15 min at room temperature under the hood.
Short vortex and spin.
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).
Stock primers and primer working solutions are stored at -20°C.
PCR
PCR
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.
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).
PCR Mix preparation :
Prepare the Master Mix :
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.
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).
Check the quality of all the PCR products on an 1.2 % agarose gel.
Figure 4 : Principle of DNA electropheris step (in French).
Safety information
Be very cautious with Ethidium Bromide manipulation!
FDS Ethidium Bromide.pdf 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)
Biorad_Gel Caster.pdf - Poor gel in the casting tray and check there are no bubbles.
- Let solidify for 20 min.
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.
Amplification result observation :
After migration, observe amplification results under UV light using an Imager (for instance : ImageQuant LAZ4000, GE Healthcare).
LAS 4000 User manual.pdf 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.
PCR product purification
PCR product purification
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.
Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf Prepare Purification Run Table and pool the triplicate PCR into a single microtube with appropriate labelling (sample, target and tag-MID nb, PCR date).
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.
PCR products quantification
PCR products quantification
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.
Quant-it Picogreen dsDNA kit.pdf 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 ).
Pooled Amplicons Concentration 4 steps
This step is performed only if the "Pooled Amplicons" has the following parameters >30µL and <35 ng/µL.
We use the kit NucleoSpin® PCR Clean-Up (Macherey-Nagel, cat. No 740609.50).
The Final Concentration is fixed at 50 ng/µL, to be sure to be in excess.
Prepare the Concentration File :
The elution volume is calculated depending on the Initial Concentration and Initial Volume of the Pooled Amplicons.
2. Add 2 *Vi µL of NT buffer and follow the recommendations of the manufacturer as mentioned in Step 9, except for the Elution Step.
Elution Step :
- Place the NucleoSpin PCR Clean-Up Column into a clean 1.5mL microtube correctly labeled (Pool Amplicon name, Quotation nb given by Fasteris, date).
- Add Vf of buffer NE (calculated in your table above) preheated at 65°C directly onto the column and incubate 1 min @ room temperature.
- Centrifuge 1 min @ 11 000 g.
Store the concentrated Pooled Amplicons at -20°C.
Pooled Amplicons Quantification and Quality Checking
Pooled Amplicons Quantification and Quality Checking
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).
MAN0019617_Qubit_1X_dsDNA_BR_Assay_UG.pdf If possible, check the final Pooled Amplicons quality on a Bioanalyzer using the kit Agilent DNA 1000 (Agilent Technologies, Cat. No 5067-1504).
G2938-90014_DNA1000Assay_KG.pdf Store the Pooled Amplicons at -80°C until the shipment to Fasteris.
Shipment Conditions to Fasteris
Shipment Conditions to Fasteris
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.