Apr 15, 2025
Whole genome amplification of human parechovirus type 3 (PEV-A3) utilizing tiling-PCR
- Nora Deezsi-Magyar1
- 1National Center for Public Health and Pharmacy
Protocol Citation: Nora Deezsi-Magyar 2025. Whole genome amplification of human parechovirus type 3 (PEV-A3) utilizing tiling-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g758q3lwz/v1
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: April 15, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 126707
Abstract
The procol allows whole genome amplification of PEV-A3, prior to next generation library preparation and sequencing. Suitable for Nanopore and Illumina sequencing as well.
DNAse I treatment (to remove contaminating human-derived gDNA)
DNAse I treatment (to remove contaminating human-derived gDNA)
40m
40m
Reagents
- TURBO DNase‱ (2 U/μL), Invitrogen
- Nuclease-free destillated water
Prepare the following master mix
| A | B | |
| TURBO DNase (2 U) | 1.0 uL | |
| TURBO DNase puffer | 4.0 uL | |
| Destillated water | 6.0 uL | |
| RNA | 40.0 uL |
Incubation in thermocycler
37 °C 00:30:00
30m
Post-DNAse I treatment clean-up
Reagents
- AMPure XP Beads, Beckman Coulter
- 70% EtOH (prepared freshly)
- Nuclease-free destillated water
Add 1.8 μl AMPure XP per 1.0 μl of sample.
Bind DNA fragments to paramagnetic beads.
Separation of beads + DNA fragments from contaminants.
Wash beads + DNA fragments twice with 70% EtOH to remove contaminants.
Elute purified DNA fragments from beads.
Transfer to new tubes.
Reverse transcription
Reverse transcription
1h 20m
1h 20m
Reagents
- SuperScript‱ VILO‱ cDNA Synthesis Kit, Invitrogen
- Nuclease-free destillated water
Prepare the following master mix
| A | B | |
| 5X VILO™ Reaction Mix | 4.0 uL | |
| 10X SuperScript™ Enzyme Mix | 2.0 uL | |
| Destillated water | 4.0 uL | |
| RNA | 8.0 uL |
Incubation in thermocycler
25 °C 00:10:00
10m
Incubation in thermocycler
42 °C 01:00:00
Incubation in thermocycler
85 °C 00:05:00
5m
Polymerase chain reaction (tiling-PCR)
Polymerase chain reaction (tiling-PCR)
2h 30m
2h 30m
Reagents
- PhusionTM High-Fidelity DNA Polymerase, New England Biolabs
- Nuclease-free destillated water
- dNTP mix
Prepare the following master mixes
Mix_1
| A | B | |
| 5x Phusion‱ High-Fidelity PCR Master Mix with GC Buffer | 4.0 uL | |
| dNTP mix (10 mM) | 0.4 uL | |
| Phusion‱ High-Fidelity DNA Polymerase | 0.2 uL | |
| Primer pool_2 | 3.0 uL | |
| Destillated water | 7.4 uL | |
| cDNA template | 5.0 uL |
The mix includes pool_1 primers (see section 4.2)
Mix_2
| A | B | |
| 5x Phusion‱ High-Fidelity PCR Master Mix with GC Buffer | 4.0 uL | |
| dNTP mix (10 mM) | 0.4 uL | |
| Phusion‱ High-Fidelity DNA Polymerase | 0.2 uL | |
| Primer pool_2 | 3.0 uL | |
| Destillated water | 7.4 uL | |
| cDNA template | 5.0 uL |
The mix includes pool_2 primers (see section 4.2)
Tiling-PCR primers
| A | B | C | D | E | |
| Primer | Pool | Sequence (5' - 3') | Size | Position (bp) | |
| hPeV-3_1_LEFT | 1 | ATACCCCGATTTGCTGAGCTTC | 22 | 41 – 987 | |
| hPeV-3_1_RIGHT | 1 | GCCATTGAATGAAAGTTATCCACATTTC | 28 | ||
| hPeV-3_2_LEFT | 2 | GATGTAGTGCAAGCTACGACCA | 22 | 899 – 1895 | |
| hPeV-3_2_RIGHT | 2 | CTACTGTGGCTGCCCAATCAAA | 22 | ||
| hPeV-3_3_LEFT | 1 | TGATCCTAGAACTGCAGGGAGT | 22 | 1750 – 2745 | |
| hPeV-3_3_RIGHT | 1 | TCTTCAGTGTCATATGTATGAGCCAC | 26 | ||
| hPeV-3_4_LEFT | 2 | AAGAGGGTCATGGCATGTTGTC | 22 | 2625 – 3631 | |
| hPeV-3_4_RIGHT | 2 | TGTAGACAAACAAGCAGTGGTTAGA | 25 | ||
| hPeV-3_5_LEFT | 1 | TCCTCAGCAGCCACAGAAATTC | 22 | 3500 – 4487 | |
| hPeV-3_5_RIGHT | 1 | TCTCTTCAAGATGTGCCATTGGG | 23 | ||
| hPeV-3_6_LEFT | 2 | GCCAGTGAGTTCATGGATGGTT | 22 | 4340 – 5339 | |
| hPeV-3_6_RIGHT | 2 | GCTGGTATCCTGCACAATGTGT | 22 | ||
| hPeV-3_7_LEFT | 1 | GCCAAACCAAAGAGTGCTTTCC | 22 | 5192 – 6188 | |
| hPeV-3_7_RIGHT | 1 | CGGACTTAACAAAGCTGTACCCT | 23 | ||
| hPeV-3_8_LEFT | 2 | GGCAAGGTGTTAAAGCATGTGTC | 23 | 6039 – 7057 | |
| hPeV-3_8_RIGHT | 2 | GCTGCTTGAATGTGCTGAAGTTT | 23 | ||
| hPeV-3_9_LEFT | 1 | ACCACCATCTTTAACACTTGTCTCA | 25 | 6296 – 7323 | |
| hPeV-3_9_RIGHT | 1 | TGGTATGTCCAATATTCCAAATTAGTGTTC | 30 |
Tiling-PCR thermocycling
1 98 °C 00:00:30
2 98 °C 00:00:10
3 54 °C 00:00:20
4 72 °C 00:00:50
5 Repeat steps 2-3-4 for 40 times.
6 72 °C 00:05:00
7 4 °C hold
6m 50s
Post PCR-clean-up
Reagents
- Ampure XP Beads, Beckman Coulter
- 70% EtOH (fershly prepared)
- Nuclease-free destillated water
Add 0.8 μl AMPure XP per 1.0 μl of sample.
Bind DNA fragments to paramagnetic beads.
Separation of beads + DNA fragments from contaminants.
Wash beads + DNA fragments twice with 70% EtOH to remove contaminants.
Elute purified DNA fragments from beads.
Transfer to new tubes.
Tiling-amplicon quantification using Qubit Flex fluorometer
Tiling-amplicon quantification using Qubit Flex fluorometer
10m
10m
Reagents
- Qubit 1X dsDNA HS Assay Kit
Add 190 uL of working solution to all wells of two Qubit strips. Label the top of the tubes S1 and S2. Then add 10 uL of the respective HS Standard (1 or 2) to each tube of the two strips.
For each of your sample tubes, add 198 uL of working solution to labeled tubes.
Then add 2 uL of your samples to each tube.
Vortex all strips and spin down if necessary. Incubate for 3-5 minutes at room temperature.
On the Qubit, select 1x dsDNA -> dsDNA High Sensitivity -> Read standards
Insert strip standard 1 and then select Read standard. Repeat for standard 2.
Select Run samples and read the respective wells of the strips. Use the 2 uL sample volume. When you are done.