TITLE: nCoV-2019 sequencing protocol for illumina
AUTHORS: Kentaro  Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Satsuki Eto, Risa Someno, Makoto Kuroda

[DESCRIPTION]
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): 
RT step has been changed to use of LunaScript (Tyson et al., 2020).
Corrected typos and wrong descriptions about amount of reagents.
Added a link to library quantification protocol.
Other minor changes.

Change histries (V4):
Added description about the primer set ver. N1/N2.
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):
Added description about a new primer set ver. N4 addressing drop-out in B.1.1.529 (Omicron).
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. 

[STEPS]
SECTION: cDNA preparation
1. 
 


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 


 

SECTION: cDNA preparation
2. 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 2 min
 55 °C for 20 min (*)
 95 °C for 1 min
Hold at  4 °C 

 

SECTION: Primer pool preparation (not everytimes)
3. 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.
 

SECTION: Primer pool preparation (not everytimes)
4. 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).

https://github.com/ItokawaK/Alt_nCov2019_primers/tree/master/Primers/ver_N4 

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.

 

 

SECTION: Multiplex PCR
5. 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
 

 

SECTION: Multiplex PCR
6. In the extraction and sample addition cabinet add 2 µL diluted cDNA to each tube and mix well by pipetting.
 

SECTION: Multiplex PCR
7. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
SECTION: Multiplex PCR
8. Set-up the following program on the thermal cycler:  

Stage                     Temperature     Time                    

Heat Activation     98 °C        30 s   

30 cycles* of ...
   Denaturation         98 °C        15 s   
   Annealing              64 °C **      5 min  
   
Hold                       4 °C            Indefinite             



 



 


SECTION: PCR clean-up
9. (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.


 


SECTION: Quantification and normalilisation (optional)
10. 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).  
SECTION: Quantification and normalilisation (optional)
11. 
 


SECTION: Fragmentation, End-prep &amp; Adapter ligation
12. 
 


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
32 °C 6 min  
65 °C 30 min 


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. 


 


Place the library plate into the thermal cycler posing at 4ºC and immediately skip to the next step (32 °C ).

SECTION: Fragmentation, End-prep &amp; Adapter ligation
13. 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.

 

 


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 15 min  
2. 65 °C 20 min  (ligase inactivation)

Start the thermal program, and place the tubes or plate immediately. 

 

SECTION: Library pooling &amp; purfication
14. 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. 
 

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.