Oct 30, 2020
Version 1
Protocol for SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing V.1
- Valeriia Kaptelova1,
- Speranskaya AS1,
- et.al1
- 1Central Research Institute of Epidemiology of the Federal Service on Customers' Rights Protection and Human Well-being Surveillance, Moscow, Russia.
- Coronavirus Method Development Community
External link: https://www.biorxiv.org/content/10.1101/2020.08.04.234880v1
Protocol Citation: Valeriia Kaptelova, Speranskaya AS, et.al 2020. Protocol for SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bn77mhrn
Manuscript citation:
bioRxiv 2020.08.04.234880; doi: https://doi.org/10.1101/2020.08.04.234880
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: October 29, 2020
Last Modified: October 30, 2020
Protocol Integer ID: 43999
Keywords: Sars-COV-2, NGS, primer panel, Whole-Genome Sequencing, COVID-19, Coronavirus Method, amplicon sequencing, Illumina library construction, coronavirus
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Abstract
Here we present a new primer panel that allows amplifying the complete genome of SARS-CoV-2 (the causative virus of COVID-19) using 17 primer pairs (in four pools). Our results demonstrate that our method allows producing full genomes when we use RNA extract from SARS-CoV-2 positive clinical samples which have a cycle threshold (Ct) in the range of 13 to 26. The resulting primer set exhibits the coverage of the entire viral genome except for only 8 bp on 5'- and 80 bp on 3'- ends in comparison with the reference genome in GenBank (accession number MT121215.1).
Guidelines
the protocol is for RESEARCH ONLY.
Materials
Protocol for SCV-2000bp a primer panel for SARS-CoV-2 full-genome sequencing.pdf Types of equipment:
- BioRad T100 thermal cycler
- Covaris M220 Ultrasonicator
- QuantStudio™ 5 Real-Time PCR System
Equipment
Bioanalyzer
NAME
Bioanalyzer
TYPE
Agilent
BRAND
G2991AA
SKU
LINK
Any bioanalyzer will suffice.
SPECIFICATIONS
- Illumina MiSeq
- Illumina HiSeq 1500
Reagents:
NEBNext End repair / dA-tailing Module (E7546)
NEBNext Ultra II Ligation Module - 96 rxnsNew England BiolabsCatalog #E7595L
Safety warnings
RNA samples should be stored at -80 °C and thawed on ice.
1. cDNA
1. cDNA
RNA samples should be stored at -80 °C and thawed on ice.
NGS library preparation and sequencing:
cDNA
Reverse transcription reaction was performed using 10 μL of the RNA samples, random hexanucleotide primers, and Reverta-L kit (AmpliSens, Russia) according to the manufacturer’s instructions.
Prepare a ready-to-use reagent mix for 12 reactions.
1.1. Add 5 µl of RT-G-mix-1 to the tube containing RT-mix,
1.2. Add 6 µl of Revertase (MMlv) into the tube with reagent mix,
1.3. Mix well
1.4. Dispense 10 µl of ready-to-use reagent mix into each prepared test tube (0.2 ml ).
1.5. Add 10 µl RNA-sample to the appropriate test tube with a ready-to-use reagent mix.
Place the test tubes into the thermocycler and incubate at 37 °С for 30 minutes.
1.6. Dilute each cDNA sample in the ratio 1:1 with DNA-buffer. To do that, add 20 µl DNAbuffer to each test tube. Carefully mix, using the pipette (10 times).
2. Amplification
2. Amplification
The cDNA was immediately used as a template for the amplification of genome fragments.
The primer panel you can find here: see Table 1.
https://www.biorxiv.org/content/10.1101/2020.08.04.234880v1.full
| Four multiplexed primer pools | cDNA | |||
| pM | μl | μl | ||
| pool 1 | 8,3 | 3 | 5 | |
| pool 2 | 10 | 2 | 8 | |
| pool 3 | 12.5 | 1 | 5 | |
| pool 4 | 10 | 2 | 5 | |
Set up the following four PCR master mixes, one for each of the four multiplexed primer pools (multiply below volumes by the number of reactions plus desired overage):
Q5 High-Fidelity DNA Polymerase was used according to the manufacturer’s instructions (New England BioLabs, NEB).
| Component | Volume per reaction (μl) | |
| Q5 master mix | 12,5 | |
| primer pool (1-4)* | variable (1-3) * | |
| H2O | 7,5 | |
| cDNA* | variable (5-8)* | |
| Total volume: | 25 |
*- It depends on the primer pool.
Amplify samples using the following PCR conditions:
| °C | Time | Cycles | |
| 98 | 0:30 s | 1 | |
| 98 | 0:10 s | 35 | |
| 64 | 0:30 s | ||
| 72 | 2:30 | ||
| 72 | 3:00 | 1 |
-20 °C SAFE STOPPING POINT
3. 1,7% agarose electrophoresis.
3. 1,7% agarose electrophoresis.
We used the following approach: at first we amplified only pool 2 because this pool works worst of all. Then products of the amplification reaction were analyzed with electrophoresis using 1,7% agarose gel stained with SYBR Green. The samples in which visible PCR products of the expected size were found were used for PCR reactions with other primer pools (1, 3, and 4). Then products of PCR were visualized using 1,7% agarose electrophoresis stained with SYBR Green.
4. mix
4. mix
Amplified fragments were mixed in equimolar amounts according to the visual estimation of concentration.
5. Clean up
5. Clean up
25m 30s
25m 30s
Clean up (in the ratio 0,7x).
Clean up PCR products of the expected size (1700-2100 bp) from the reaction mixture and to remove the nonspecific short fragments obtained during the amplification step.
Premixed amplicons were cleaned in the ratio 0,7x using Agencourt AMPure XP (Beckman Coulter, Danvers, MA, USA)
Incubate at Room temperature for 00:05:00
5m
Place the tubes on a magnet: allow beads to separate for 00:05:00
5m
Remove supernatant.
Add 200 µl 80% fresh ethanol to wash the beads, incubate for00:00:30
30s
Remove 200 µl 80% EtOH.
Repeat steps 5.5-5.6.
Briefly spin the tubes and remove additional ethanol. Ensure any visible quantities of ethanol are removed.
Leave the beads in open tubes for 00:05:00
5m
Add low TE and incubate for 00:05:00 .
5m
Place the tubes on a magnet: allow beads to separate for 00:05:00
5m
Transfer supernatant into the new tubes.
SAFE STOPPING POINT!Store at -20 °C.
6. Qubit
6. Qubit
Qubit dsDNA HS Assay Kit.
Prepare the Qubit® working solution by diluting the Qubit® dsDNA HS Reagent 1:200 in Qubit® dsDNA HS Buffer. Use a clean plastic tube each time you prepare a Qubit® working solution.
Add Qubit® working solution to individual assay tubes so that the final volume in each tube after adding a sample is 200 µL.
Note: Your sample can be anywhere from 1–20 µL (We use 1µL). Add a corresponding volume of Qubit® working solution to each assay tube: anywhere from 180–199 µL.
7. Covaris.
7. Covaris.
Covaris.
PCR products (50 µl)were sheared in microTUBE-50 AFA Fiber Screw-Cap (PN 520166) using Covaris M220 (Covaris, Woburn, MA) using the following settings: peak incident power (W)- 75, duty factor- 5%, cycles per burst-200, treatment Time (s) - 50, temperature (°C)-20, sample volume (μl)- 50.
8. Clean up
8. Clean up
Clean up (in the ratio 1,5x)
See point 5.2-5.12
9. Qubit
9. Qubit
Qubit dsDNA HS Assay Kit.
See point 6.
10. End repair with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
10. End repair with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
1h
1h
End repair with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
Starting Material: 11-100 ng in total reaction.
Mix the following components in a sterile, nuclease-free tube:
| COMPONENT VOLUME | VOLUME, µL, PER REACTION | |
| (green) NEBNext Ultra II End Prep Enzyme Mix | 3 | |
| (green) NEBNext Ultra II End Prep Reaction Buffer | 7 | |
| Fragmented DNA (11-100 ng in total reaction) | variable (1-5 µL) | |
| nuclease free water | variable (45- 49 µL) | |
| Total Volume | 60 |
Set a 100 μl or 200 μl pipette to 50 μl and then gently pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:
20 °C for 00:30:00
65 °C for 00:30:00
Hold at4 °C
1h
11. Ligation with NEBNext Ultra II Ligation Module (E7595)
11. Ligation with NEBNext Ultra II Ligation Module (E7595)
15m
15m
Ligation with NEBNext Ultra II Ligation Module (E7595) and Y-shaped adapters compatible with Nextera XT Index Kit.
need to dilute adaptor to 3 pM (for DNA input was 11-100 ng in total reaction) (0,6 pM for 1-10 ng in total reaction)
Add the following components directly to the End Prep reaction mixture (60 µl) and mix well:
| COMPONENT VOLUME | VOLUME, µL, PER REACTION | |
| Y-shaped adapters compatible with Nextera XT Index Kit (dilution adapter) | 2,5 | |
| (red) NEBNext Ligation Enhancer | 1 | |
| (red) NEBNext Ultra II Ligation Master Mix | 30 | |
| Total Volume | 93,5 |
Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours at 4°C. We do not recommend premixing the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.
Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
(Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).
Incubate at20 °C for 00:15:00 in a thermocycler with the heated lid off.
15m
12. Clean up
12. Clean up
Clean up (in the ratio 1,5x)
See point 5.2-5.12
13. Indexing PCR
13. Indexing PCR
Indexing PCR
| COMPONENT VOLUME | VOLUME, µL, PER REACTION | |
| DNA | 8,5 | |
| Q5 Master Mix | 12,5 | |
| Index /i5 Primer (5pM) | 2 | |
| Index /i7 Primer (5pM) | 2 | |
| Total (mix) | 25 |
* for Real-time PCR used 1µL EvaGreen (20x).
Amplify samples using the following PCR conditions:
| °C | Time | Cycles | |
| 98 | 0:30 s | 1 | |
| 98 | 0:10 s | 8 | |
| 65 (detect) | 1:15 m |
* for low-concentration samples used 9-10 cycles.
14. Clean up
14. Clean up
Clean up (in the ratio 1,2x)
See point 5.2-5.12
15. Agilent 2100 Bioanalyzer.
15. Agilent 2100 Bioanalyzer.
Agilent 2100 Bioanalyzer.
The quality and fragment length distribution of the obtained libraries were evaluated with Agilent Bioanalyzer 2100 (Agilent Technologies, USA).
16. Size selection (300-600bp).
16. Size selection (300-600bp).
30m 30s
30m 30s
Size selection (300-600bp).
The size selection of the final libraries was done using Agencourt SPRISelect Reagent (Beckman Coulter, Danvers, MA, USA).
Selecting for DNA larger than a target size.
The size selection protocol is based on a starting volume of 100 μl. Adjust the final volume by adding nuclease-free water for a 100 μl total volume.
Add 60 μl of resuspended AMPure XP beads to the 100 μl. Mix well by pipetting up and down at least 10 times.
Incubate for 00:05:00 at Room temperature
5m
Place the tube on an appropriate magnetic stand to separate the beads from the supernatant.After the solution is clear (about 5 minutes), carefully transfer the supernatant containing your DNA to a new tube (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.
SPRIselect Right Workflow
Selecting for DNA smaller than a target size or selecting for DNA within a target region.
Add 20 μl resuspended AMPure XP beads to the supernatant, mix well and incubate for 00:05:00 at Room temperature
5m
Place the tubes on a magnet: allow beads to separate for 00:05:00
5m
Remove supernatant.
Add 200 µl80% fresh ethanol to wash the beads, incubate for 00:00:30
30s
Remove 200 µl 80% EtOH.
Repeat steps 5.5-5.6.
Briefly spin the tubes and remove additional ethanol. Ensure any visible quantities of ethanol are removed.
. Leave the beads in open tubes for 00:05:00
5m
Add low TE and incubate for 00:05:00 .
5m
Place the tubes on a magnet: allow beads to separate for 00:05:00
5m
Transfer supernatant into the new tubes.
17. Agilent 2100 Bioanalyzer.
17. Agilent 2100 Bioanalyzer.
Agilent 2100 Bioanalyzer.
See point 15.
18. Quantitation of next-generation sequencing (NGS) libraries (NEBNext® Library Quant Kit for Illumina)
18. Quantitation of next-generation sequencing (NGS) libraries (NEBNext® Library Quant Kit for Illumina)
Quantitation of next-generation sequencing (NGS) libraries (NEBNext® Library Quant Kit for Illumina)
The NEBNext® Library Quant Kit has been optimized to provide substantial performance and workflow improvements to qPCR-based library quantitation.
qPCR was performed according to the manufacturer’s instructions.
HiSeq or MiSeq
HiSeq or MiSeq
Sequencing was performed on Illumina HiSeq 1500 with HiSeq PE Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2 (500 cycles) or Illumina MiSeqwith the MiSeq Reagent Kit V2 (500 cycles) or V3 (600 cycles) according to the manufacturer’s instructions.
For HiSeq Rapid Run v2 dilute the pooled sample to8 picomolar (pM) in HT1, following HiSeq 1500 loading instructions. For Miseq v3 dilute the pooled sample to 12 picomolar (pM) in HT1. For Miseq v2 dilute the pooled sample to 8 picomolar (pM) in HT1, following MiSeq loading instructions.