Preparation of viral sequencing library for Illumina using WTA2 and QIAseq FX

Collect 400µL virus culture medium in a 1.5 mL tube.

Centrifuge 0h 3m 0s at 17,000 x g and aspirate the supernatant with a 1 mL tuberculin syringe.

Equipment

Value	Label
New Steradisc	NAME
0.45μm filter 50pcs	TYPE
Kurabo	BRAND
S-1304	SKU
https://www.kurabo.co.jp/english/	LINK

Filter the medium through a 0.45μm filter into a 1.5 mL tube.

Mix the following reagents in a new 1.5mL tube.

   **Component                       Volume / sample** 



Micrococcal nuclease   `1µL`



Benzonase                     `2µL`



Homemade buffer*       `7µL` 

*see MATERIALS

Add 200µL of filtrate into the tube, then mix by pipetting.

Incubate at 37°C for 2h 0m 0s.

Extract RNA from total volume (210µL) and elute to 50µL.

Prepare 2.5µL of template nucleic acid in an 0.2mL 8-strip tube on ice.

Note

This protocol uses 1/5 reagents per sample compared to the original WTA2 kit.

Add the following components in the tube.

  **Component                                      Volume / sample** 



 Nuclease-free water                `0.32µL`     (possible to be replaced by template nucleic acid)



 Synthesis solution (WTA2)      `0.5µL` 

Total so far: 3.32µL

Mix and incubate the reaction as follows:

1. 95°C for 0h 5m 0s

2. Hold at 18°C

Set the thermal cycler with a program below and start.

1. 18°C pose

2. 18°C for 0h 10m 0s

3. 25°C for 0h 10m 0s

4. 37°C for 0h 30m 0s

5. 42°C for 0h 10m 0s

6. 70°C for 0h 20m 0s

7. Hold at 4°C

Mix the following components, keep at 18°C, and add to the template from step 10.

**Component                                                   Volume / sample** 



 Library Synthesis Buffer (WTA2)       `0.5µL`



 Nuclease-free water                          `0.78µL`



 Library Synthesis Enzyme (WTA2)    `0.4µL` 

Total so far: 5µL

Transfer the reaction tubes on the thermal cycler kept at 18°C, and immediately skip to the next step (18°C for 0h 10m 0s).

Mix the following components as master mix.

 **Component                                           Volume / sample** 



 Nuclease-free water                    `60.2µL`



 Amplification Mix (WTA2)             `7.5µL`



 WTA dNTP Mix (WTA2)                 `1.5µL`



 Amplification Enzyme (WTA2)      `0.75µL`

Add the master mix to the Library Synthesis reaction from step 13.

Total so far: approximately 75µL

Transfer the reaction tubes on the thermal cycler.

Set the thermal cycler with a program below and start.

1. 94°C for 0h 2m 0s

2. 20 cycles x (94°C for 0h 0m 30s, 70°C for 0h 5m 0s)

3. Hold at 4°C

Clean-up the amplicons using

Add 90µL of AMpure XP per sample.(Mixing ratio that removes below 100 bp)

Incubate atRoom temperature for 0h 5m 0s

Separate magnetic beads and remove supernatant.

To wash beads, add 150µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (1/2)

To wash beads, add 150µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (2/2)

Allow the beads to dry for 0h 2m 0s.

Elute purified amplicon in 37.5µL of Nuclease-free water.

Quantify the purified amplicon using fluorescent based method using

.

Concentrations in the range of 10-100 ng/µL of purified amplicon are sufficient for the next section.

The use of

or

is assumed in this protocol.

Note

This protocol uses 1/8 reagents per sample compared to the original QIAseq FX DNA library kit.

32°C

Set the thermal cycler with a program below and start.

Keep the heat-lid at 80°C.

1. 32°C pose

2. 32°C 0h 8m 0s

3. 65°C 0h 30m 0s

Place new 8-strip tubes at 96 well aluminum block On ice.

Prepare a reaction mix per one sample as below.

**Component                                   Volume / sample** 

FX Buffer, 10x 0.625µL

FX Enzyme Mix 1.25µL

Purified amplicon Use liquid volume equivalent to between 20 to 100 ng.

Nuclease-free water up to 4.375µL

Total 6.25µL

Transfer the tubes from the ice to the thermal cycler, and immediately skip to the next step (32°C).

Add 0.5µL adapter solution to each end-prepped DNA mixture.

Prepare a master mix per sample below On ice .

Component Volume / sample

DNA Ligase Buffer, 5x 2.5µL

DNA Ligase 1.25µL

Nuclease-free water 2µL

Total 5.75µL

Add 5.75µL of above master mix to each end-prepped DNA mixture mixed with adapterOn ice.

Total so far: 12.5µL

Set a thermal cycler with the following program with heat lid at 80°C.

1. 20°C 0h 15m 0s

2. 65°C 0h 20m 0s

Place the tubes, and start the thermal program immediately.

Take the ligated mixture from each well and pool them into the 1.5 mL low-binding tube.

Adjust the volume to be pooled to average the amount of DNA in each sample.

Briefly measure the volume of pooled mixture using pipette.

Clean-up the pooled library using

Add AMpure XP to library using x0.8 volume of the libary (Mixing ratio that removes below 150 bp)

Incubate atRoom temperature for 0h 5m 0s.

Separate magnetic beads and remove supernatant.

To wash beads, add 500µL of 80% ethanol, and mix.

Separate magnetic beads and remove supernatant.(1/2)

To wash beads, add 500µL of 80% ethanol, and mix.

Separate magnetic beads and remove supernatant.(2/2)

Allow the beads to dry for 0h 2m 0s.

Elute DNA in 50µL of nuclease-free water.

Transfer the eluted DNA to a new 1.5 mL low-binding tube.

Purify again by adding 60µL of AMpure XP (x1.2 volume of the elution which allow to remove below 100 bp ).

Incubate atRoom temperature for 0h 5m 0s.

Separate magnetic beads and remove supernatant.

To wash beads, add 500µL of 80% ethanol, and mix.

Separate magnetic beads and remove supernatant.(1/2)

To wash beads, add 500µL of 80% ethanol, and mix.

Separate magnetic beads and remove supernatant.(2/2)

Allow the beads to dry for 0h 2m 0s.

Finally, elute DNA in 30µL low-TE (10mM Tris-HCl pH8.0, 0.1mM EDTA).

Transfer the eluted DNA to a new 1.5 mL low-binding tube.

Quantify the purified library using

Mix 5µL of the library with loading dye and electrophoresis on a 2% agarose gel alongside molecular markers.

Obtain a smear image of the library.

Estimate approximate average library size (base pairs) on the smear image.

The size of the most concentrated region can be read and used as an estimate.

Calculate molar concentration of the library using the formula below.

Y (nM) = X (ng/µL) ÷Z (base pairs) ÷ 660 (g/mol) ×10⁶

Y: molar concentration of the library

X: mass concentration of the library

Z: average library size

Dilute the library to 1 nM using resuspension buffer of PhiX Control.

Prepare final library mixture as below.

Components volume

Resuspension buffer 93µL

PhiX control (50 pM ) 2µL

Library (1 nM) 5µL