TITLE: Preparation of viral sequencing library for Illumina using WTA2 and QIAseq FX
AUTHORS: Kenichi Komabayashi

[DESCRIPTION]
This method uses a metagenomic approach to analyze the genome sequence of DNA and RNA viruses. Nucleic acids outside the viral particles are reduced using nucleases and extracted to obtain template DNA and RNA. Templates are converted to double-stranded DNA by random amplification, and library preparation is performed for analysis on Illumina sequencers.
Analysis data with reduced sequences of host and bacterial origin and abundant sequences of viral origin are obtained, allowing multiple samples to be analyzed even with the throughput of the iSeq100.

The library preparation protocol was originally folked from "nCoV-2019 sequencing protocol for illumina protocol V5" by Itokawa et al.



[GUIDELINES]
There are three advantages to using this method.
(1)No need to design virus-specific primers
(2)Applicable to both DNA and RNA viral genomes
(3)10 or more samples can be analyzed at a time on the iSeq100 (For genome analysis of coxsackievirus A6)

The method consists of three parts: pretreatment, random amplification, and library preparation.

The pretreatment is intended to increase the content of virus-derived nucleic acids in the sample and facilitate genome analysis. The main point of this method is to reduce host genome, ribosomal RNA, and nucleic acids derived from bacteria in advance, taking advantage of the fact that genomes in viral particles are not easily digested by Nuclease.

The random amplification using Merck millipore sigma's WTA2 kit can be used to obtain double-stranded DNA amplicon using DNA and RNA as templates. The following three points are different from the method described in the attached manual.
(1) This protocol is performed at one-fifth the scale of the protocol described in the manual.
(2) The initial denaturation temperature is changed so that DNA is also used as a template.
(3) The number of cycles of PCR amplification is increased due to the lower initial nucleic acid content.

The library preparation protocol was originally folked from "nCoV-2019 sequencing protocol for Illumina protocol V5" by Itokawa et al. Since the QIAseq FX DNA Library kit is used for library preparation in this method, multiplex analysis with the library of SARS-Cov-2 genome sequencing obtained using the protocol by Itokawa et al.


[STEPS]
SECTION: Reduction of nucleic acids derived from non-virus
1. Collect 400 µL virus culture medium in a 1.5 mL tube.

 

SECTION: Reduction of nucleic acids derived from non-virus
2. Centrifuge 3 min at 17,000 x g  and aspirate the supernatant with a 1 mL tuberculin syringe.
SECTION: Reduction of nucleic acids derived from non-virus
3. 
 

Filter the medium through a 0.45μm filter into a 1.5 mL tube.
SECTION: Reduction of nucleic acids derived from non-virus
4.   
  

Mix the following reagents in a new 1.5mL tube.
        Component                       Volume / sample
	Micrococcal nuclease   1 µL
	Benzonase                     2 µL
	Homemade buffer*       7 µL 
*see MATERIALS
SECTION: Reduction of nucleic acids derived from non-virus
5. Add 200 µL of filtrate into the tube, then mix by pipetting.
SECTION: Reduction of nucleic acids derived from non-virus
6. Incubate at 37 °C for 120 min.
SECTION: Reduction of nucleic acids derived from non-virus
7.   

Extract RNA from total volume (210 µL) and elute to 50 µL.
SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
9. 
  

 

Add the following components in the tube.
     Component                                      Volume / sample
     Nuclease-free water                0.32 µL     (possible to be replaced by template nucleic acid)
     Synthesis solution (WTA2)      0.5 µL 

Total so far: 3.32 µL 
SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
11. Set the thermal cycler with a program below and start. 

  1.   18 °C pose
  2.  18 °C for 10 min
  3.  25 °C for 10 min
  4.  37 °C for 30 min
  5.  42 °C for 10 min
  6.  70 °C for 20 min
  7.  Hold at 4 °C
SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
14. Mix the following components as master mix.
     Component                                           Volume / sample
     Nuclease-free water                    60.2 µL
     Amplification Mix (WTA2)             7.5 µL
     WTA dNTP Mix (WTA2)                 1.5 µL
     Amplification Enzyme (WTA2)      0.75 µL

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

Total so far: approximately 75 µL
SECTION: PCR clean-up and quantification
16. Clean-up the amplicons using   

Add 90 µL of AMpure XP per sample.(Mixing ratio that removes below 100 bp)
SECTION: PCR clean-up and quantification
23. 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.

SECTION: Fragmentation, End-prep &amp; Adapter ligation
24. The use of 
  or
  
is assumed in this protocol.

 

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   8 min   
  3. 65 °C   30 min   
SECTION: Fragmentation, End-prep &amp; Adapter ligation
29. Prepare a master mix per sample below on ice .

  Component                                    Volume / sample
   DNA Ligase Buffer, 5x            2.5 µL   
   DNA Ligase                             1.25 µL   
   Nuclease-free water              2 µL  
   Total                                        5.75 µL

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

Total so far: 12.5 µL
SECTION: Library pooling &amp; purfication
31. 
 
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.
SECTION: Library pooling &amp; purfication
33. Incubate atRoom temperature for 5 min.
SECTION: Preparation of 50pM library for Illumina iSeq100
47. Quantify the purified library using   

SECTION: Preparation of 50pM library for Illumina iSeq100
49. 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.


 

SECTION: Preparation of 50pM library for Illumina iSeq100
50. 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

 

SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
8. Prepare 2.5 µL of template nucleic acid in an 0.2mL 8-strip tube on ice.
SECTION: Preparation of 50pM library for Illumina iSeq100
48. Mix 5 µL of the library with loading dye and electrophoresis on a 2% agarose gel alongside molecular markers.

Obtain a smear image of the library.
SECTION: Preparation of 50pM library for Illumina iSeq100
51.   

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

Prepare final library mixture as below.
Components                              volume
Resuspension buffer            93 µL
PhiX control (50 pM )           2 µL
Library (1 nM)                       5 µL

SECTION: Fragmentation, End-prep &amp; Adapter ligation
28. Add 0.5 µL adapter solution to each end-prepped DNA mixture.
SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
10. Mix and incubate the reaction as follows:
   1.  95 °C   for 5 min
   2.  Hold at   18 °C 
SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
12. Mix the following components, keep at 18°C, and add to the template from step 10.
     Component                                                   Volume / sample
     Library Synthesis Buffer (WTA2)       0.5 µL
     Nuclease-free water                          0.78 µL
     Library Synthesis Enzyme (WTA2)    0.4 µL 

Total so far: 5 µL
SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
13. Transfer the reaction tubes on the thermal cycler kept at 18 °C, and immediately skip to the next step (18 °C for 10 min).

SECTION: Whole transcriptome amplification independent of 3&#x27; end sequence
15. Transfer the reaction tubes on the thermal cycler.

Set the thermal cycler with a program below and start.
   1.  94 °C for 2 min
   2.  20 cycles x (94 °C for 30 s, 70 °C for 5 min)
   3.  Hold at 4 °C
SECTION: PCR clean-up and quantification
18. Separate magnetic beads and remove supernatant.
SECTION: PCR clean-up and quantification
19. To wash beads, add 150 µL of 80% ethanol, incubate for 30 s, and remove supernatant (1/2)
SECTION: PCR clean-up and quantification
20. To wash beads, add 150 µL of 80% ethanol, incubate for 30 s, and remove supernatant (2/2)
SECTION: PCR clean-up and quantification
21. Allow the beads to dry for 2 min.
SECTION: PCR clean-up and quantification
22. Elute purified amplicon in 37.5 µL of Nuclease-free water.
SECTION: PCR clean-up and quantification
17. Incubate atRoom temperature for 5 min
SECTION: Fragmentation, End-prep &amp; Adapter ligation
25. Place new 8-strip tubes at 96 well aluminum block on ice.
SECTION: Fragmentation, End-prep &amp; Adapter ligation
26. Prepare a reaction mix per one sample as below.

   Component                                   Volume / sample
   FX Buffer, 10x                          0.625 µL   
   FX Enzyme Mix                        1.25 µL 
   Purified amplicon                    Use liquid volume equivalent to between 20 to 100 ng.
   Nuclease-free water      up to 4.375 µL
   Total                                          6.25 µL 
SECTION: Fragmentation, End-prep &amp; Adapter ligation
27. Transfer the tubes from the ice to the thermal cycler, and immediately skip to the next step (32 °C).
SECTION: Fragmentation, End-prep &amp; Adapter ligation
30. Set a thermal cycler with the following program with heat lid at 80 °C.
  1.  20 °C  15 min    
  2. 65 °C  20 min 

Place the tubes, and start the thermal program immediately. 
SECTION: Library pooling &amp; purfication
34. Separate magnetic beads and remove supernatant.
SECTION: Library pooling &amp; purfication
35. To wash beads, add 500 µL of 80% ethanol, and mix.
Separate magnetic beads and remove supernatant.(1/2)
SECTION: Library pooling &amp; purfication
37. Allow the beads to dry for 2 min.
SECTION: Library pooling &amp; purfication
38. Elute DNA in 50 µL of nuclease-free water.
SECTION: Library pooling &amp; purfication
39. Transfer the eluted DNA to a new 1.5 mL low-binding tube.
SECTION: Library pooling &amp; purfication
40. Purify again by adding 60 µL of AMpure XP (x1.2 volume of the elution which allow to remove below 100 bp ).
SECTION: Library pooling &amp; purfication
32. 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)
SECTION: Library pooling &amp; purfication
36. To wash beads, add 500 µL of 80% ethanol, and mix.
Separate magnetic beads and remove supernatant.(2/2)
SECTION: Library pooling &amp; purfication
41. Incubate atRoom temperature for 5 min.
SECTION: Library pooling &amp; purfication
42. Separate magnetic beads and remove supernatant.
SECTION: Library pooling &amp; purfication
43. To wash beads, add 500 µL of 80% ethanol, and mix.
Separate magnetic beads and remove supernatant.(1/2)
SECTION: Library pooling &amp; purfication
44. To wash beads, add 500 µL of 80% ethanol, and mix.
Separate magnetic beads and remove supernatant.(2/2)
SECTION: Library pooling &amp; purfication
45. Allow the beads to dry for 2 min.
SECTION: Library pooling &amp; purfication
46. Finally, elute DNA in 30 µL low-TE (10mM Tris-HCl pH8.0, 0.1mM EDTA).

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