TITLE: Preparation of viral sequencing library for Illumina using NEBNext ultra II
AUTHORS: Kenichi Komabayashi

[DESCRIPTION]
This method uses a metagenomic approach to analyze the genome sequence of RNA viruses. Nucleic acids outside the viral particles are reduced using nucleases and extracted to obtain template RNA. Templates are converted to double-stranded DNA, 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.

This protocol was folked from "Preparation of viral sequencing library for Illumina using WTA2 and QIAseq FX".


[GUIDELINES]
There are three advantages to using this method.
(1)No need to design virus-specific primers
(2)RNA viral genomes can be analyzed while reducing nucleotides of host and bacterial origin
(3)10 or more samples can be analyzed at a time on the iSeq100 (For viral genome less than 10,000 bases)

The method consists of two parts: pretreatment 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.

NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770) is used for library preparation.
The following points are different from the method described in the attached manual.
(1) Use half the scale of the protocol in the manual.
(2) The number of cycles of PCR amplification is increased because the initial RNA input is small.
The amplified libraries are pooled into a tube for multiplex analysis. The size and molar concentration of the pooled libraries are adjusted to obtain a final library that can be applied to the flow cell.


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

 

SECTION: Reduction of nucleic acids derived from non-virus (pretreatment)
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 (pretreatment)
3. 
 

Filter the medium through a 0.45μm filter into a 1.5 mL tube.
SECTION: Reduction of nucleic acids derived from non-virus (pretreatment)
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 (pretreatment)
5. Add 200 µL of filtrate into the tube, then mix by pipetting.
SECTION: Reduction of nucleic acids derived from non-virus (pretreatment)
6. Incubate at 37 °C for 120 min.
SECTION: Reduction of nucleic acids derived from non-virus (pretreatment)
7.   

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

SECTION: End Prep of cDNA Library
23. Mix the following components, keepon ice 
     Component                                                            Volume / sample
     Product from step 13                                        25 µL
     End Prep Reaction Buffer (kit E7770)              3.5 µL
     End Prep Reaction Enzyme (kit E7770)           1.5 µL

Total so far: 30 µL
SECTION: Clean-up using magnetic beads
15. Clean-up products using   

Add 72 µL (1.8x) of AMpure XP per sample.
SECTION: End Prep of cDNA Library
24. Incubate in a thermal cycler set with the following program.
Keep the heat-lid at 75 °C.  
      1.  20 °C for 30 min
      2. 65 °C for 30 min
      3.  Hold at 4 °C
SECTION: Clean-up using magnetic beads
17. Separate magnetic beads and remove supernatant.
SECTION: Clean-up using magnetic beads
18. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (1/2)
SECTION: Clean-up using magnetic beads
19. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (2/2)
SECTION: Clean-up using magnetic beads
20. Allow the beads to dry for 2 min.
SECTION: Clean-up using magnetic beads
21. Elute purified product in 26 µL of 0.1x TE (kit E7770).
SECTION: Clean-up using magnetic beads
16. Incubate atRoom temperature for 5 min
SECTION: Fragmentaion of RNA and priming
8. 0.2 mL PCR tubes are used to incubate mixtures.
For steps 9 to 47, refer to section 2 'Protocol for use with NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat)' in a manual of NEBNext Ultra II RNA Library Prep kit for Illumina (kit E7770). In our protocol, section 2.5 and beyond is referred.
Half volume of the reagent listed in the manual is used. 
SECTION: Fragmentaion of RNA and priming
9. Mix the following components, keepon ice
     Component                                                                 Volume / sample
     RNA                                                                      2.5 µL
     First Strand Synthesis Buffer (kit E7770)          2.0 µL
     Random Primers (kit E7770)                               0.5 µL 

Total so far: 5 µL
SECTION: Fragmentaion of RNA and priming
10. Incubate in a thermal cycler set with the following program.
Keep the heat-lid at 105 °C.  
     1.94 °C   for 10 min
     2.  Hold at   4 °C 
SECTION: Synthesis of 2nd strand cDNA
13. Mix the following components, keepon ice 
      Component                                                                        Volume / sample
     Product from step 13                                                10 µL
     Second Strand Synthesis Buffer (kit E7770)           4.0 µL
     Second Strand Synthesis Enzyme (kit E7770)        2.0 µL
     Nuclease Free Water                                                 24 µL

Total so far: 40 µL
SECTION: Synthesis of 1st strand cDNA
11. Mix the following components, keepon ice 
      Component                                                                Volume / sample
     Product from step 10                                          5.0 µL
     First Strand Sysnthesis Enzyme (kit E7770)     1.0 µL
     Nuclease Free Water                                          4.0 µL

Total so far: 10 µL
SECTION: Synthesis of 1st strand cDNA
12. Incubate in a thermal cycler set with the following program.
Keep the heat-lid at 80 °C.  
      1.  25 °C for 10 min
      2.  42 °C for 50 min
      3.  70 °C for 15 min
      4.  Hold at 4 °C
SECTION: Synthesis of 2nd strand cDNA
14. Incubate in a thermal cycler set with the following program.
Keep the heat-lid at 40 °C.  
     1.16 °C   for 60 min
     2.  Hold at   4 °C 
SECTION: Clean-up using magnetic beads
22. Separate magnetic beads and transfer  25 µL of supernatant to a new 0.2 mL tube.
SECTION: Adaptor ligation
25. Mix the following components in a 1.5 mL low-binding tube, keep on ice 
      Component                                                                                         Volume
     NEBNext Adaptor for Illumina (E7335 or E7500)          1.0 µL
     Adaptor Dilution Buffer(kit E7770)                                 199 µL
SECTION: Adaptor ligation
26. Mix the following components as master mix in a 1.5 mL tube, keep on ice 
      Component                                                Volume / sample
     Ligation Enhancer (kit E7770)           0.5 µL
     Ligation Master Mix (kit E7770)        15 µL
SECTION: Adaptor ligation
27. Mix the following components, in the order given, keep on ice 
      Component                                                Volume / sample
     Product from step 24                         30 µL
     Diluted Adaptor (step 25)                  1.25 µL
     Master mix (step 26)                          15.5 µL

Total so far: 46.75 µL
SECTION: Adaptor ligation
28. Incubate in a thermal cycler set with the following program.
Keep the heat-lid at 45 °C.  
      1.  20 °C for 15 min
      2.  20 °C pose *
      3.  37 °C for 15 min
      4.  Hold at 15 °C
      
* Add USER Enzyme (kit E7770)  1.5 µL / sample and mix

Total so far: 48.25 µL
SECTION: Clean-up using magnetic beads
29. Clean-up products using   

Add 43.5 µL (0.9x) of AMpure XP per sample.
SECTION: Clean-up using magnetic beads
30. Incubate atRoom temperature for 10 min
SECTION: Clean-up using magnetic beads
31. Separate magnetic beads and remove supernatant.
SECTION: Clean-up using magnetic beads
32. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (1/2)
SECTION: Clean-up using magnetic beads
33. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (2/2)
SECTION: Clean-up using magnetic beads
34. Allow the beads to dry for 2 min.
SECTION: Clean-up using magnetic beads
35. Elute purified product in 8.0 µL of 0.1x TE (kit E7770).
SECTION: Clean-up using magnetic beads
36. Separate magnetic beads and transfer  7.5 µL of supernatant to a new 0.2 mL tube.
SECTION: PCR Enrichment of Adaptor Ligated DNA
37. Mix the following components, keep on ice 
      Component                                                                                Volume / sample
     Adaptor Ligated DNA from step 36                        7.5 µL 
     Q5 Master Mix (kit E7770)                                       12.5 µL 
     Index (X) Primer (E7335 or E7500)                         2.5 µL 
     Universal PCR Primer (E7335 or E7500)                2.5 µL 

For multiplex analysis of specimens fewer than seven, use 'Index oligo selector'  to verify that the index combination is acceptable.
NEBNext Index oligo selector
SECTION: PCR Enrichment of Adaptor Ligated DNA
38. Incubate in a thermal cycler set with the following program.

   1.  98 °C for 30 s
   2.  20 cycles x (98 °C for 10 s, 65 °C for 75 s)
   3.  Hold at 4 °C
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
39. Clean-up products using   

Add 22.5 µL (0.9x) of AMpure XP per sample.
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
40. Incubate atRoom temperature for 5 min
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
41. Separate magnetic beads and remove supernatant.
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
42. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (1/2)
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
43. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (2/2)
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
44. Allow the beads to dry for 2 min.
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
45. Elute purified product in 11.5 µL of 0.1x TE (kit E7770).
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
46. Separate magnetic beads and transfer  11 µL of supernatant to a new 0.2 mL tube.
SECTION: Clean-up of PCR product using magnetic beads and quantification of DNA
47. 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: Library pooling
48. Take the purified PCR product from each tube 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.

Add 0.1x TE (kit E7770) up to 50 µLof total volume.  

SECTION: Estimation of library size
58. Quantify the purified library using   


 

SECTION: Estimation of library size
59. 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: Estimation of library size
60. 
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 estimation.


 

SECTION: Preparation of 50pM library for Illumina iSeq100
61. 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: Purification of the library for size selection
49. Clean-up products using   

Add 30 µL (0.6x) of AMpure XP per sample.
SECTION: Purification of the library for size selection
50. Incubate atRoom temperature for 5 min
SECTION: Purification of the library for size selection
51. Separate magnetic beads and remove supernatant.
SECTION: Purification of the library for size selection
52. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (1/2)
SECTION: Purification of the library for size selection
53. To wash beads, add 200 µL of 80% ethanol, incubate for 30 s, and remove supernatant (2/2)
SECTION: Purification of the library for size selection
54. Allow the beads to dry for 2 min.
SECTION: Purification of the library for size selection
55. Elute purified product in 30 µL of 0.1x TE (kit E7770).
SECTION: Purification of the library for size selection
56. Separate magnetic beads and transfer supernatant to a new 0.2 mL tube.
SECTION: Purification of the library for size selection
57. Quantify the purified amplicon using fluorescent based method using   . 
Concentrations 1.5 ng/µL or more of purified library is sufficient for the next section.

SECTION: Setting up the local run manager in iSeq100
62. To analyze libraries using NEBNext‱ multiplex oligos for Illumina, you need to load the index and other information into a local run manager.

Refer to the manuals on Illumina site
How to use a custom library prep kit in Local Run Manager v2
How to use custom library prep and index kits with Local Run Manager v3 and v4

Obtain a .tsv file for configuration from the NEBNext‱ multiplex oligo page on NEB.

Change DefaultReadLength1, 2 in the .tsv file from 251 to 151.

Start the Local Run Manager on iseq100 and open "Tools" on the dashboard.

In the drop-down menu, select "Index & Library Prep Kits", "Index Kit", and "Add Index Kit".

Select and load the modified .tsv file.