General fungi ITS2 (ITS4ngsUni - fITS7) for Illumina amplicon sequencing
Roey Angel, Eva Petrova
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.
Before start
Steps
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
Prepare the following master mixture 0On 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 |
Vortex and spin down 0h 2m 0s .
Distribute 24µL of the mixture to each tube or well of 96-well plate and add 1µL of template DNA or cDNA.
PCR program
Run the following PCR program:
1. 95°C 0h 10m 0s
2. x 30 {
a. 95°C 0h 0m 45s
b. 52°C 0h 0m 45s
c. 72°C``0h 0m 45s
}
4. 72°C 0h 10m 0s
10°C
Evaluate PCR products on an agarose gel
Prepare a 1.5% agarose gel by mixing:
100mL TAE
1.5g agarose
Heat in the microwave until dissolved and pour into a gel frame.
Place solid gel into an electrophoresis bath filled with TAE buffer.
Load 5µL of the sample into a well.
In addition load 5µL of DNA ladder mix (80-10,000 bp) into an empty well, as a marker.
Run the gel at 110 V, 265 mA for approx.0h 40m 0s
Stain gel for at least 0h 30m 0s in an Ethidium bromide TAE bath (or any other DNA stain).
Visualize the gel using a gel documentation system.
