Preparation of viral sequencing library for Illumina using NEBNext ultra II

Kenichi Komabayashi

Published: 2024-07-22 DOI: 10.17504/protocols.io.kqdg3xrw1g25/v1

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Abstract

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".

Steps

Reduction of nucleic acids derived from non-virus (pretreatment)

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

Note

If the viral particles are broken, the genome could be digested in this later process.

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.

Note

If the concentration of RNA is measured here using the Qubit RNA HS Assay Kit or similar, it is not measurable due to low concentration. You can understand the large amount of viral genes are still included using real time PCR etc.

Fragmentaion of RNA and priming

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.

Mix the following components, keep4On ice

**Component                                                                 Volume / sample** 



 RNA                                                                      `2.5µL`



 First Strand Synthesis Buffer (kit E7770)          `2.0µL`



 Random Primers (kit E7770)                               `0.5µL`

Total so far: 5µL

10.

Incubate in a thermal cycler set with the following program.

Keep the heat-lid at 105°C.

 1.`94°C`   for `0h 10m 0s`



 2.  Hold at   `4°C`

Synthesis of 1st strand cDNA

11.

Mix the following components, keepOn ice

Component Volume / sample

 Product from step 10                                          `5.0µL`



 First Strand Sysnthesis Enzyme (kit E7770)     `1.0µL`



 Nuclease Free Water                                          `4.0µL`

Total so far: 10µL

12.

Incubate in a thermal cycler set with the following program.

Keep the heat-lid at 80°C.

  1.  `25°C` for `0h 10m 0s`



  2.  `42°C` for `0h 50m 0s`



  3.  `70°C` for `0h 15m 0s`



  4.  Hold at `4°C`

Synthesis of 2nd strand cDNA

13.

Mix the following components, keepOn ice

Component Volume / sample

 Product from step 13                                                `10µL`



 Second Strand Synthesis Buffer (kit E7770)           `4.0µL`



 Second Strand Synthesis Enzyme (kit E7770)        `2.0µL`



 Nuclease Free Water                                                 `24µL`

Total so far: 40µL

14.

Incubate in a thermal cycler set with the following program.

Keep the heat-lid at 40°C.

 1.`16°C`   for `1h 0m 0s`



 2.  Hold at   `4°C`

Clean-up using magnetic beads

15.

Clean-up products using

Add 72µL (1.8x) of AMpure XP per sample.

16.

Incubate atRoom temperature for 0h 5m 0s

17.

Separate magnetic beads and remove supernatant.

18.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (1/2)

19.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (2/2)

20.

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

21.

Elute purified product in 26µL of 0.1x TE (kit E7770).

22.

Separate magnetic beads and transfer 25µL of supernatant to a new 0.2 mL tube.

End Prep of cDNA Library

23.

Mix the following components, keepOn ice

 **Component                                                            Volume / sample** 



 Product from step 13                                        `25µL`



 End Prep Reaction Buffer (kit E7770)              `3.5µL`



 End Prep Reaction Enzyme (kit E7770)           `1.5µL`

Total so far: 30µL

24.

Incubate in a thermal cycler set with the following program.

Keep the heat-lid at 75°C.

  1.  `20°C` for `0h 30m 0s`



  2. `65°C` for `0h 30m 0s`



  3.  Hold at `4°C`

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)          `1.0µL`



 Adaptor Dilution Buffer(kit E7770)                                 `199µL`

26.

Mix the following components as master mix in a 1.5 mL tube, keep On ice

Component Volume / sample

 Ligation Enhancer (kit E7770)           `0.5µL`



 Ligation Master Mix (kit E7770)        `15µL`

27.

Mix the following components, in the order given, keep On ice

Component Volume / sample

 Product from step 24                         `30µL`



 Diluted Adaptor (step 25)                  `1.25µL`



 Master mix (step 26)                          `15.5µL`

Total so far: 46.75µL

28.

Incubate in a thermal cycler set with the following program.

Keep the heat-lid at 45°C.

  1.  `20°C` for `0h 15m 0s`



  2.  `20°C` pose *



  3.  `37°C` for `0h 15m 0s`



  4.  Hold at `15°C`

Add USER Enzyme (kit E7770) 1.5µL / sample and mix

Total so far: 48.25µL

29.

Clean-up products using

Add 43.5µL (0.9x) of AMpure XP per sample.

30.

Incubate atRoom temperature for 0h 10m 0s

31.

Separate magnetic beads and remove supernatant.

32.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (1/2)

33.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (2/2)

34.

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

35.

Elute purified product in 8.0µL of 0.1x TE (kit E7770).

36.

Separate magnetic beads and transfer 7.5µL of supernatant to a new 0.2 mL tube.

PCR Enrichment of Adaptor Ligated DNA

37.

Mix the following components, keep On ice

Component Volume / sample

 Adaptor Ligated DNA from step 36                        `7.5µL` 



 Q5 Master Mix (kit E7770)                                       `12.5µL` 



 Index (X) Primer (E7335 or E7500)                         `2.5µL` 



 Universal PCR Primer (E7335 or E7500)                `2.5µL`

For multiplex analysis of specimens fewer than seven, use 'Index oligo selector' to verify that the index combination is acceptable.

NEBNext Index oligo selector

38.

Incubate in a thermal cycler set with the following program.

98°C for 0h 0m 30s
20 cycles x (98°C for 0h 0m 10s, 65°C for 0h 1m 15s)
Hold at 4°C

Clean-up of PCR product using magnetic beads and quantification of DNA

39.

Clean-up products using

Add 22.5µL (0.9x) of AMpure XP per sample.

40.

Incubate atRoom temperature for 0h 5m 0s

41.

Separate magnetic beads and remove supernatant.

42.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (1/2)

43.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (2/2)

44.

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

45.

Elute purified product in 11.5µL of 0.1x TE (kit E7770).

46.

Separate magnetic beads and transfer 11µL of supernatant to a new 0.2 mL tube.

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.

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.

Note

For example, if you want to obtain 100 ng from each sample, 2µL of the 50 ng/µL sample and 4µL of the 25 ng/µL sample should be aliquoted. In the next section, purification with 0.6x AMPureXP to remove DNA of less than 300 bp resulted in the loss of much DNA (reduced to about one-eighth of the amount). Therefore, if the total amount of DNA obtained in this section is small (approximately 500 ng / 50 µL or less), it will be difficult to obtain a visible agarose gel electrophoresis in the section on library size estimation.If the total amount of DNA from library pooling is small, removal of less than 300 bp of DNA should not be performed.

Briefly measure the volume of pooled mixture using pipette.

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

Purification of the library for size selection

49.

Clean-up products using

Add 30µL (0.6x) of AMpure XP per sample.

50.

Incubate atRoom temperature for 0h 5m 0s

51.

Separate magnetic beads and remove supernatant.

52.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (1/2)

53.

To wash beads, add 200µL of 80% ethanol, incubate for 0h 0m 30s, and remove supernatant (2/2)

54.

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

55.

Elute purified product in 30µL of 0.1x TE (kit E7770).

56.

Separate magnetic beads and transfer supernatant to a new 0.2 mL tube.

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.

Estimation of library size

58.

Quantify the purified library using

Note

More accurate molar concentrations can be determined using commercially available library quantification kits instead of the methods shown in this section.

59.

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.

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.

Note

Image J is helpful to recognize distribution of the library size. You can obtain a densitogram of the gel image. https://imagej.net/ij/

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

Note

See the Illumina website.'Converting ng/µl to nM when calculating dsDNA library concentration'https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-reference_material-list/000001240

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.

Note

If libraries are created using both E7335 and E7500, a .tsv file that has both index information can be used.You can consolidate the information under [Indices] in the two .tsv files into one file, and change the "Name" and "Description" under [kit] as desired.

Preparation of viral sequencing library for Illumina using NEBNext ultra II

Disclaimer

Abstract

Steps

Reduction of nucleic acids derived from non-virus (pretreatment)

Fragmentaion of RNA and priming

Synthesis of 1st strand cDNA

Synthesis of 2nd strand cDNA

Clean-up using magnetic beads

End Prep of cDNA Library

Adaptor ligation

PCR Enrichment of Adaptor Ligated DNA

Clean-up of PCR product using magnetic beads and quantification of DNA

Library pooling

Purification of the library for size selection

Estimation of library size

Preparation of 50pM library for Illumina iSeq100

Setting up the local run manager in iSeq100

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