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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was originally folked from "</span><a href="https://dx.doi.org/10.17504/protocols.io.bbmuik6w" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">ARTIC amplicon sequencing protocol for MinION for nCoV-2019</span></a><span>" by Josh Quick</span><span style = "font-weight:bold;"> </span><span>to adapt for</span><span style = "font-weight:bold;"> </span><span>any illumina sequencers (iSeq, MiSeq, NextSeq, etc.)</span><span style = "font-weight:bold;">.</span><span>  </span></div><div class = "text-block">  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. </div><div class = "text-block">  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.</div><div class = "text-block"><span style = "font-weight:bold;">Change histories (V2)</span><span>: </span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">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. </li><li style = "counter-reset:ol0;">Amount of adapter solution input was corrected (Step 19).</li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">Change histories (V3)</span><span>: </span></li><li style = "counter-reset:ol0;"><span>RT step has been changed to use of LunaScript (</span><a href="https://doi.org/10.1101/2020.09.04.283077" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Tyson et al., 2020</span></a><span>).</span></li><li style = "counter-reset:ol0;">Corrected typos and wrong descriptions about amount of reagents.</li><li style = "counter-reset:ol0;">Added a link to library quantification protocol.</li><li style = "counter-reset:ol0;">Other minor changes.</li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">Change histries ( V4):</span></li><li style = "counter-reset:ol0;"><span>Added description about the primer set ver. </span><a href="https://github.com/ItokawaK/Alt_nCov2019_primers/tree/master/Primers" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">N1/N2</span></a><span style = ":;">.</span></li><li style = "counter-reset:ol0;">Added suggestion to double the concentration of nCoV-2019_74_LEFT and nCoV-2019_74_RIGHT.</li><li style = "counter-reset:ol0;">Default annealing & extension  temperature of multiplex PCR was changed to 64 °C. </li><li style = "counter-reset:ol0;">Optional normalization step after PCR clean-up was omitted.</li></ol></div><div class = "text-block"><span style = "font-weight:bold;">Change histories (V3)</span><span>: </span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>RT step has been changed to use of LunaScript (</span><a href="https://doi.org/10.1101/2020.09.04.283077" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Tyson et al., 2020</span></a><span>).</span></li><li style = "counter-reset:ol0;">Corrected typos and wrong descriptions about amount of reagents.</li><li style = "counter-reset:ol0;">Added a link to library quantification protocol.</li><li style = "counter-reset:ol0;">Other minor changes.</li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">Change histries ( V4):</span></li><li style = "counter-reset:ol0;"><span>Added description about the primer set ver. </span><a href="https://github.com/ItokawaK/Alt_nCov2019_primers/tree/master/Primers" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">N1/N2</span></a><span style = ":;">.</span></li><li style = "counter-reset:ol0;">Added suggestion to double the concentration of nCoV-2019_74_LEFT and nCoV-2019_74_RIGHT.</li><li style = "counter-reset:ol0;">Default annealing & extension  temperature of multiplex PCR was changed to 64 °C. </li><li style = "counter-reset:ol0;">Optional normalization step after PCR clean-up was omitted.</li></ol></div><div class = "text-block"><span style = "font-weight:bold;">Change histries ( V4):</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>Added description about the primer set ver. </span><a href="https://github.com/ItokawaK/Alt_nCov2019_primers/tree/master/Primers" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">N1/N2</span></a><span style = ":;">.</span></li><li style = "counter-reset:ol0;">Added suggestion to double the concentration of nCoV-2019_74_LEFT and nCoV-2019_74_RIGHT.</li><li style = "counter-reset:ol0;">Default annealing & extension  temperature of multiplex PCR was changed to 64 °C. </li><li style = "counter-reset:ol0;">Optional normalization step after PCR clean-up was omitted.</li></ol></div></div>

[STEPS]
?. [cDNA preparation]
Mix the following components in an 0.2mL 8-strip tube or 96 well PCR plate;Component                                        VolumeLunaScript RT SuperMix                  Template RNA  (purified)                 Total
This protocol uses 5/8  reagents per sample  compared to the original LunaScript protocol.
1.25 µl
5 µl
6.25 µl
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.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.Incubate the reaction as follows: for  for  (*) for Hold at
25 °C
55 °C
95 °C
4 °C
*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.
?. [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.
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [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                           dNTPs (10 mM each)                            Q5 Hot Start DNA Polymerase             Primer Pool 1 or 2 (50 µM)                   Nuclease-free water                              Total
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
0.72 µl
0.72 µl
16.53 µl
16.53 µl
23 µl
23 µl
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.
?. [Multiplex PCR]
In the extraction and sample addition cabinet add  diluted cDNA to each tube and mix well by pipetting.
2 µl
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Multiplex PCR]
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Stage                     Temperature     Time                    Heat Activation     30 cycles* of ...   Denaturation            Annealing               **      Hold                                   Indefinite
98 °C
98 °C
64 °C
4 °C
*) 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.
**) 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    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.
?. [PCR 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.
?. [PCR clean-up]
Clean-up the amplicons using the AmpureXP using 1x volume. Elute in  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.
20 µl
Amplicon clean-up 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)]
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).
?. [Quantification and normalilisation (optional)]
!!!  We do not recommend normalizing library concentrations at this step but after adapter ligation. It is more immune to sample swapping. !!!
* In our experience, samples with DNA concentration less than   in 30 cycles  at this point do not generate meaningful results.
You can expect the quantity of DNA input will be directly proportional to the amount of data you will obtain if no further adjustment takes place.
?. [Fragmentation, End-prep & Adapter ligation]
Transfer   of pooled & purified 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   .Set the thermal cycler with a program below and start. Keep the heat-lid at  .Prepare a master mix per one sample below.Component                         Volume / sampleFX Buffer, 10x                      H2O                                       FX Enzyme Mix                   Total                                     Add    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 ( ).
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).
2 µl
on ice
80 °C
[ pose]
32 °C
65 °C
1.25 µl
6.75 µl
2.5 µl
10.5 µl
10.5 µl
Make sure the plate is always    during this procedure to avoid fragmentation reaction proceeds at this step.
32 °C
?. [Fragmentation, End-prep & Adapter ligation]
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  adapter solution to each end-prepped DNA mixture.Prepare a master mix per sample below.Component                            Volume / sampleDNA Ligase Buffer, 5x           DNA Ligase                             H2O                                          Total                                        Add  of above master mix to each end-prepped DNA mixture mixed with adapter  .Set a thermal cycler with the following program with heat lid at  .1. 2.   (ligase inactivation)Start the thermal program, and place the tubes or plate immediately.
Keep both end-prepped DNA mixture and thawed adapter solution     during this step.
1 µl
5 µl
2.5 µl
4 µl
11.5 µl
11.5 µl
on ice
80 °C
20 °C
65 °C
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]
Prepare a 1.5 or 2.0 ml low-binding tube.Take * ligated mixture from each well and pool them into the 1.5 or 2.0 ml low-binding tube. Finaly, elute DNA in    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    low-TE or Elution Buffer.Transfer the eluted DNA to a new 1.5 or 2.0 ml low-binding tube.
5 µl
*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.
50 µl
25 µl
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.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.
?. [Primer pool preparation (not everytimes)]
About N1/N2 primer setSince March 2021, we have added another small modification on the N1 primer set  (ver. N2). The modification is addition of one primer "nCoV-2019_72_RIGHT_C22A" to pool2 mixture. This primer contains an alternative nucleotide corresponding to G22017T mutation seen in R.1 (B.1.1.316.1) lineage to prevent amplicon 72 dropout. nCoV-2019_1_LEFT5 µLnCoV-2019_1_RIGHTv25 µLnCoV-2019_3_LEFT5 µL......nCoV-2019_97_LEFT5 µLnCoV-2019_97_RIGHT5 µLTotal490 µLHere is the recommended formulation of N2 primer set.nCoV-2019_1_LEFT5 µLnCoV-2019_1_RIGHTv25 µLnCoV-2019_3_LEFT5 µL......nCoV-2019_97_LEFT5 µLnCoV-2019_97_RIGHT5 µLTotal490 µLnCoV-2019_2_LEFT5 µLnCoV-2019_2_RIGHT5 µL......nCoV-2019_72_LEFT5 µLnCoV-2019_72_RIHGT5 µLnCoV-2019_72_RIGHT_C22A5 µLnCoV-2019_74_LEFT10 µLnCoV-2019_74_RIGHT10 µL......nCoV-2019_98_LEFT5 µLnCoV-2019_98_RIGHT5 µLTotal505 µL
nCoV-2019_1_LEFT5 µLnCoV-2019_1_RIGHTv25 µLnCoV-2019_3_LEFT5 µL......nCoV-2019_97_LEFT5 µLnCoV-2019_97_RIGHT5 µLTotal490 µL
nCoV-2019_1_LEFT5 µLnCoV-2019_1_RIGHTv25 µLnCoV-2019_3_LEFT5 µL......nCoV-2019_97_LEFT5 µLnCoV-2019_97_RIGHT5 µLTotal490 µL
nCoV-2019_2_LEFT5 µLnCoV-2019_2_RIGHT5 µL......nCoV-2019_72_LEFT5 µLnCoV-2019_72_RIHGT5 µLnCoV-2019_72_RIGHT_C22A5 µLnCoV-2019_74_LEFT10 µLnCoV-2019_74_RIGHT10 µL......nCoV-2019_98_LEFT5 µLnCoV-2019_98_RIGHT5 µLTotal505 µL