Feb 06, 2024
General fungi ITS2 (ITS4ngsUni - fITS7) for Illumina amplicon sequencing
- 1Soil and Water Research Infrastructure
Protocol Citation: Roey Angel, Eva Petrova 2024. General fungi ITS2 (ITS4ngsUni - fITS7) for Illumina amplicon sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6em5gqe5/v1
Manuscript citation:
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: February 08, 2018
Last Modified: February 06, 2024
Protocol Integer ID: 10124
Abstract
A general assay for the preparation of PCR amplicons for further analysis of fungal communities by Illumina amplicon sequencing.
The primers target the 2nd internal transcribed spacer region (ITS2) of the ribosomal RNA gene and were specifically designed for Illumina amplicon sequencing. The ITS4ngsUni primer was designed by Tedersoo et al. (2016) as a universal primer for nearly all eukaryotes, and the fITS7 primer was designed by Ihrmark et al. (2012) as a fungal-specific primer. The ITS2 region primer pair should capture a higher diversity compared to primers targeting the ITS1 region.
For barcoding, we use the Fludigm Access Array, and therefore, the primers are synthesized with the CS1 and CS2 regions.
When working with soil samples, we usually obtain amplicons in the range of 300-500bp. Double bands are often observed.
Protocol materials
AgaroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9539
Step 6
GeneRuler DNA Ladder MixThermo Fisher ScientificCatalog #SM0331
In 2 steps
DreamTaq DNA Polymerase (5 U/µL)Thermo FisherCatalog #EP0701
Step 2
0.2 mM dNTPsThermo Fisher ScientificCatalog #AM8200
Step 2
Bovine Serum Albumin (BSA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
Step 2
Primers
Primers
| A | B | C | D | |
| Name | Sequnce | Ref. | Target region 1 | |
| ITS4ngsUni_CS1 | ACA CTG ACG ACA TGG TTC TAC ACG CCT SCS CTT ANT DAT ATG C | Tedersoo et al. (2016) | 5.8S | |
| fITS7_CS2 | TAC GGT AGC AGA GAC TTG GTC TGG GTG ART CAT CGA ATC TTT G | Ihrmark et al. (2012) | ITS-flanking site in LSU |
1 within the ribosomal RNA gene
PCR reaction
PCR reaction
Prepare the following master mixture On ice .
Do not forget to prepare some additional mixture for the negative (NTC = no template) and positive controls, and to account for pipetting errors. Work in a clean PCR box.
| A | B | C | D | |
| Reagent | Final. conc. | 1 tube(25μl) μl | 96 tubes (25μl x100) | |
| PCR H2O | 16,775 | 1677,5 | ||
| 10X DreamTaq Green Buffer | 1X | 2,5 ul | 250 | |
| dNTP (2 mM each) | 0.2 mM | 2,5 | 250 | |
| BSA (20 μg μl-1) | 80 ng µl-1 | 0,1 | 10 | |
| ITS4ngsUni-CS1 linker | 0.5 μM | 1 | 100 | |
| fITS7-CS2 linker | 0.5 μM | 1 | 100 | |
| DreamTaq Green DNA Polymerase | 0.625 U | 0,125 | 12,5 | |
| Final volume | 25 | 2500 |
DreamTaq DNA Polymerase (5 U/µL)Thermo FisherCatalog #EP0701
0.2 mM dNTPsThermo Fisher ScientificCatalog #AM8200
Bovine Serum Albumin (BSA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
Vortex and spin down 00:02:00 .
2m
Distribute 24 µL of the mixture to each tube or well of 96-well plate and add 1 µL of template DNA or cDNA.
PCR program
PCR program
Run the following PCR program:
1. 95 °C 00:10:00
2. x 30{
a. 95 °C 00:00:45
b. 52 °C 00:00:45
c. 72 °C 00:00:45
}
4. 72 °C 00:10:00
5. 10 °C 00:00:00 hold
2h
Evaluate PCR products on an agarose gel
Evaluate PCR products on an agarose gel
1h 10m
Prepare a 1.5% agarose gel by mixing:
100 mL TAE
1.5 g agarose
Heat in the microwave until dissolved and pour into a gel frame.
Place solid gel into an electrophoresis bath filled with TAE buffer.
AgaroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9539
GeneRuler DNA Ladder MixThermo Fisher ScientificCatalog #SM0331
GeneRuler DNA Ladder MixThermo Fisher ScientificCatalog #SM0331
1h
Load 5 µL of the sample into a well.
In addition load 5 µL of DNA ladder mix (80-10,000 bp) into an empty well, as a marker.
10m
Run the gel at 110 V, 265 mA for approx.00:40:00
40m
Stain gel for at least 00:30:00 in an Ethidium bromide TAE bath (or any other DNA stain).
Safety information
Ethidium bromide is not regulated as hazardous waste at low concentrations, but is treated as hazardous waste by many organizations. Material should be handled according to the manufacturer's Safety Data Sheet (SDS).
Most use of ethidium bromide in the laboratory (0.25–1 µg/ml) is below the LD50 dosage, making acute toxicity unlikely. Testing in humans and longer studies in a mammalian system would be required to fully understand the long-term risk ethidium bromide poses to lab workers, but it is clear that ethidium bromide can cause mutations in mammalian and bacterial cells.
30m
Visualize the gel using a gel documentation system.
Protocol references
Tedersoo, L. et al. Tree diversity and species identity effects on soil fungi, protists and animals are context dependent. ISME J. 10, 346–362 (2016).
Ihrmark, K. et al. New primers to amplify the fungal ITS2 region - evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiol. Ecol. 82, 666–677 (2012).