Dec 06, 2021
Version 5
nCoV-2019 sequencing protocol for illumina V.5
Version 1 is forked from nCoV-2019 sequencing protocol
- Kentaro Itokawa1,
- Tsuyoshi Sekizuka1,
- Masanori Hashino1,
- Rina Tanaka1,
- Satsuki Eto1,
- Risa Someno1,
- Makoto Kuroda1
- 1National Institute of Infectious Diseases, Japan
Protocol Citation: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Satsuki Eto, Risa Someno, Makoto Kuroda 2021. nCoV-2019 sequencing protocol for illumina. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly398mgx9/v5Version created by Kentaro Itokawa
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: December 05, 2021
Last Modified: July 14, 2022
Protocol Integer ID: 55698
Keywords: SARS-CoV-2, Genome Sequencing, illumina, B.1.1.529, B.1.617.2, PrimalSeq,
Abstract
This protocol was originally folked from "ARTIC amplicon sequencing protocol for MinION for nCoV-2019" by Josh Quick to adapt for any illumina sequencers (iSeq, MiSeq, NextSeq, etc.).
Because the PCR products are fragmented and ligated with adapters, this protocol is compatible to many sequencing kits with various read lengths (75PE, 150PE, etc.) for Illumina machines.
While the library preparation uses QIAseq FX by Qiagen and is basically straight forward (as par kit instruction but modified to 1/4 scale), some tweaks for much of simplicity and speed were added.
Change histories (V2):
- The amount of cDNA input to multiplex PCR is increased (now almost same amount to the ARTIC Network's original protocol) (Step 10). This change gives better results for samples with extremely low RNA copy.
- Amount of adapter solution input was corrected (Step 19).
Change histories (V3):
- Corrected typos and wrong descriptions about amount of reagents.
- Added a link to library quantification protocol.
- Other minor changes.
Change histries (V4):
- Added suggestion to double the concentration of nCoV-2019_74_LEFT and nCoV-2019_74_RIGHT.
- Default annealing & extension temperature of multiplex PCR was changed to 64 °C.
- Optional normalization step after PCR clean-up was omitted.
Change histries (V5):
- Clean-up of each PCR products became optional. Instead, you can just dilute them and go.
- PCR volume was reduced to 1/2.
Guidelines
The important issue you have to consider first is how many samples you will multiplex in single run. This all depends on the capacity of your sequencer and the sample's viral loads represented by Ct-values in qPCR clinical test.
If a sample contains a relatively high copy number of virus genome (say, Ct < 25), the obtained reads usually distribute evenly across the genome. In such cases, only 10 Mb (330x) per sample is enough to cover the whole genome with sufficient coverage (>30x).
On the other hand, coverage bias increases as a sample's Ct-value increases. Hence, you will need more data to recover relatively weak regions. For samples containing only a low copy number of virus genome (Ct ~ 32), at least 100 Mb (3300x) is desirable to sequence the most part of the genome.
Also, if you are going to multiplex many samples in a low-throughput model (e.g. iSeq100), intensive normalization for each sample indix will be needed.
Materials
STEP MATERIALS
QIAseq FX DNA Library KitQiagenCatalog #180475
QIAseq FX DNA Library KitQiagenCatalog #180475
Protocol materials
QIAseq FX DNA Library KitQiagenCatalog #180475
QIAseq FX DNA Library KitQiagenCatalog #180475
QIAseq FX DNA Library KitQiagenCatalog #180475
cDNA preparation
cDNA preparation
Note
This protocol uses 5/8 reagents per sample compared to the original LunaScript protocol.
Mix the following components in an 0.2mL 8-strip tube or 96 well PCR plate;
Component Volume
LunaScript RT SuperMix 1.25 µL
Template RNA (purified) 5.0 µL
Total 6.25 µL
Note
A mastermix should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
Incubate the reaction as follows:
25 °C for 00:02:00
55 °C for 00:20:00 (*)
95 °C for 00:01:00
Hold at 4 °C
Note
*Incubation for 20 min on 55 °C is used instead of 10 min of the kit protocol to make sure cDNA is synthesized efficiently. However, we have not confirmed the benefit of this modification, yet.
23m
Primer pool preparation (not everytimes)
Primer pool preparation (not everytimes)
If concentration of your primer stocks are 50 µM, generate primer pool stocks by adding each primers equally or as described in tables below to Eppendorf tubes labelled either “Pool 1 (50 µM)” or “Pool 2 (50 µM)”. These are your 50 µM stocks of each primer pool.
If concentration of the primer stocks are 100 µM, dilute this primer pool 1:1 in molecular grade water, to generate 50 µM primer stocks.
It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
Note
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
About primer set
This protocol may be comaptible to any primer schemes (ARTIC Network V3/4, MidNight, NEB VerSkip, etc..).
N1 (NIID ver.1) primer set is a modified version of the ARTIC Network's V1 primer set. The N1 primer set includes replacement of 12 primers which involved in dimer formation with other primers (Itokawa et al., 2020).
Since December 2021, we updated the primer set to ver. N4. The modifications include additional primers addressing amplicon drop-out in two lineage of concern (VOC), B.1.617.2 (Delta) and B.1.1.529 (Omicron).
Also, we confirmed that doubling the concentrations of
nCoV-2019_64_LEFT, nCoV-2019_64_RIGHT, nCoV-2019_74_LEFT and nCoV-2019_74_RIGHT primers improves inherent low coverage of amplicon 64 and 74.
Here is the recommended formulation of N2 primer set.
| Primer | Volume | |
| nCoV-2019_1_LEFT | 5 µL | |
| nCoV-2019_1_RIGHTv2 | 5 µL | |
| nCoV-2019_3_LEFT | 5 µL | |
| ... | ... | |
| nCoV-2019_97_LEFT | 5 µL | |
| nCoV-2019_97_RIGHT | 5 µL | |
| nCoV-2019_73_LEFT_b11529b | 5 µL | |
| nCoV-2019_75_RIGHT_b11529a | 5 µL | |
| nCoV-2019_83_LEFT_b11529a | 5 µL | |
| Total | 505 µL |
Example of primer mixing for Pool1 of the N4 primer set
| Primer | Volume | |
| nCoV-2019_2_LEFT | 5 µL | |
| nCoV-2019_2_RIGHT | 5 µL | |
| ... | ... | |
| nCoV-2019_64_LEFT | 10 µL | |
| nCoV-2019_64_RIHGT | 10 µL | |
| ... | ... | |
| nCoV-2019_74_LEFT | 10 µL | |
| nCoV-2019_74_RIGHT | 10 µL | |
| ... | ... | |
| nCoV-2019_98_LEFT | 5 µL | |
| nCoV-2019_98_RIGHT | 5 µL | |
| nCoV-2019_72_RIGHT_b16172a | 5 µL | |
| nCoV-2019_76_LEFT_b11529a | 5 µL | |
| nCoV-2019_92_LEFT_b11529a | 5 µL | |
| Total | 525 µL |
Example of primer mixing for Pool2 of the N4 primer set
Multiplex PCR
Multiplex PCR
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2 mL 8-strip PCR tubes or 96-well PCR plate:
Component Pool 1 Pool 2
5X Q5 Reaction Buffer 2.5 µL 2.5 µL
dNTPs (10 mM each) 0.25 µL 0.25 µL
Q5 Hot Start DNA Polymerase 0.125 µL 0.125 µL
Primer Pool 1 or 2 (50 µM) 0.36 µL 0.36 µL
Nuclease-free water 7.265 µL 7.265 µL
Total 10.5 µL 10.5 µL
Note
A PCR mastermix for each pool should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
Note
If you use Q5 HS Hi-Fi 2X Master Mix, use 61-62 °C for Annealing & Extention temparature for following PCR.
In the extraction and sample addition cabinet add 2 µL diluted cDNA to each tube and mix well by pipetting.
Note
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
Set-up the following program on the thermal cycler:
Stage Temperature Time
Heat Activation 98 °C 00:00:30
30 cycles* of ...
Denaturation 98 °C 00:00:15
Annealing 64 °C ** 00:05:00
Hold 4 °C Indefinite
Note
*) We recommend 30 PCR cycles regardless of Ct-value because comparing PCR product yield and Ct-value gives a simple safeguard against sample swapping. However, one could increase this number to 35 to achieve more even PCR product yields.
Note
**) Previously, 65 °C was used for the annealing/extension step in multiplex PCR. However, inspecting many results in different thermocyclers and institutes, we now consider setting this temperature to 64 °C rather than 65 °C consistently yields better result with N1/N2 primer set. Nevertheless, fine-tuning of this temperature may still recommended to obtain the best result. Generally, amplicon 64 tends to show low coverage if annealing/extension temparature is higher than optimal.
Temperature and coverage of amplicons with N1 primer set and the protocol version 1 in TaKaRa Thermal Cycler Dice Touch. Modified from Itokawa et al., 2020 (CC BY 4.0).
PCR clean-up
PCR clean-up
(Option 1: With clean-up)
Combine each 10 µl contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a new 8-strip PCR tubes or 96-well PCR plate.
Clean-up the amplicons using the AmpureXP using 1x volume.
Elute in 20 µL of low-TE buffer (10 mM tris-HCl pH8.0, 0.1 mM EDTA).
Transfer eluted DNA to a new 8-strip PCR tubes or 96-well PCR plate.
(Option 2: No clean-up)
Just combine each 2 µL contents of “Pool 1” and “Pool 2” PCR reactions with 16 µL milli-Q water (x5 dilution) for each biological sample.
Note
Amplicon clean-up or dilution should be performed in the post-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
Quantification and normalilisation (optional)
Quantification and normalilisation (optional)
Quantify the amplicon pools using fluorescent based method such as Qubit dsDNA HS kit (Invitrogen), Qaunt-it High-Sensitivity dsDNA Assay Kit (Invitrogen), or QuantiFluor® dsDNA System (Promega).
Note
* In our experience, samples with DNA concentration less than 2 Mass Percent in 30 cycles at this point do not generate meaningful results.
Fragmentation, End-prep & Adapter ligation
Fragmentation, End-prep & Adapter ligation
Note
This protocol uses 1/4 reagents per sample compared to the original QIAseq FX DNA library kit (Qiagen: 180473 for 24-plex, 180475 and 180477 for 96-plex, and 180479, 180480, 180481 and 180482 for 384-plex).
Transfer 2 µL of pooled & purified (diluted) PCR products to each well of a new 8-strip PCR tubes or 96-well PCR plate. Then, place the tubes or plate at 96 well aluminum block On ice .
Set the thermal cycler with a program below and start. Keep the heat-lid at 80 °C .
4 °C pose
32 °C 00:06:00
65 °C 00:30:00
Prepare a master mix per one sample below.
Component Volume / sample
FX Buffer, 10x 1.25 µL
H2O 6.75 µL
FX Enzyme Mix 2.5 µL
Total 10.5 µL
Add 10.5 µL of the above master mix to each well of the 8-strip PCR tubes or 96-well PCR plate. Mix well by pipetting.
Note
Make sure the plate is always On ice during this procedure to avoid fragmentation reaction proceeds at this step.
Place the library plate into the thermal cycler posing at 4ºC and immediately skip to the next step (32 °C ).
Remove the tube or plate from the thermal cycler after finishing the thermal program.
Take the adapter plate out of the kit box and thaw the content.
QIAseq FX DNA Library KitQiagenCatalog #180475
Note
Keep both end-prepped DNA mixture and thawed adapter solution On ice during this step.
Add 1 µL adapter solution to each end-prepped DNA mixture.
Prepare a master mix per sample below.
Component Volume / sample
DNA Ligase Buffer, 5x 5 µL
DNA Ligase 2.5 µL
H2O 4 µL
Total 11.5 µL
Add 11.5 µL of above master mix to each end-prepped DNA mixture mixed with adapter On ice .
Set a thermal cycler with the following program with heat lid at 80 °C .
1. 20 °C 00:15:00
2. 65 °C 00:20:00 (ligase inactivation)
Start the thermal program, and place the tubes or plate immediately.
Note
Seal and store remaining adapter plate in -20 °C. Extreme care should be taken to avoid cross-contamination between different indexed adapters.
Library pooling & purfication
Library pooling & purfication
Prepare a 1.5 or 2.0 ml low-binding tube.
Take 5 µL * ligated mixture from each well and pool them into the 1.5 or 2.0 ml low-binding tube.
Note
*You can take different volumes by sample to adjust the DNA quantities for even representation of each index See the (Guidelines & Warnings). Use the DNA concentrations measured after the PCR clean-up.
Briefly measure the volume of pooled mixture in the 1.5 ml or 2.0 ml low-binding tube.
Purify by Ampure XP using x0.8 volume.
Finaly, elute DNA in 50 µL low-TE or Elution Buffer.
Transfer the eluted DNA to a new 1.5 or 2.0 ml low-binding tube.
Purify again by Ampure XP using x1.2 volume.
Finaly, elute DNA in 25 µL low-TE or Elution Buffer.
Transfer the eluted DNA to a new 1.5 or 2.0 ml low-binding tube.
Note
Now, the library is ready for sequencing after quantification.
You may use any your routine methods for library quantification.
We recommend qPCR based methods in terms of accuracy and sensitivity.
Here is our homemade protocol.
Protocol
NAME
Illumina TruSeq Library quantification with qPCR probe method
CREATED BY
Kentaro Itokawa
If you do not have a good quantification technique, here is a relationship between DNA mass concentration measured by Qubit dsDNA HS kit (Invitrogen) versus molar concentration measured by qPCR for nine libraries we obtained with this protocol.
Library DNA mass concentration measured by QuBit versus molar concentration measured by qPCR