TITLE: SARS-CoV-2 DNA library preparation using an adapted version of Illumina DNA prep protocol v.1.1
AUTHORS: Emmanuel  Kagning Tsinda, Masahiro Sakamoto, Michiko Okamoto, Clyde  Dapat, Mariko Saito, Mayuko Saito, Hitoshi Oshitani

[DESCRIPTION]
This protocol describes the steps to prepare DNA libraries from PCR amplicons using the Illumina DNA prep library kit. The current library preparation protocol was adapted from the original Illumina DNA Prep Reference Guide (document #1000000025416 v09) using low input DNA samples as the starting material.  
We omitted laboratory equipment such as the Eppendorf 96-well PCR plate, microseal adhesive seals.  In addition, we replaced 96-well plate magnetic stand with a magnetic stand suitable for 1.5 ml tubes. 
The added value of this protocol is that PCR reactions happen in 0.2 ml PCR tubes, and it can be implemented without a separate purchase of 96-well PCR plates or a magnetic stand for PCR plates.  Using individual 0.2 ml tubes increases the user flexibility when running few samples for library preparation. The other advantage of using our adapted protocol is the reduced library preparation cost when running few specimens. For instance, implementing the current protocol might be cheaper than the original Illumina protocol when using the Illumina® DNA Prep, (M) Tagmentation (24 Samples) catalog number 20018704. 
The protocol has proven effective for processing hundreds of DNA libraries from tiled virus amplicons such as Sapovirus and SARS-CoV-2 submitted to public repositories such as GISAID and GenBank.

[BEFORE_START]
Please follow steps 1 to 25 of the protocol below ARTIC protocol below.
 

After the multiplex PCR using the ARTIC protocol (https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgxjjxkn),  please pool P1 and P2 amplicons into a single 1.5 ml tube; then clean-up the amplicons using magnetic beads following the manufacturer's protocol. 
Quantify the cleaned DNA product Qubit DNA High-sensitivity kit (important). 
Dilute the genomic DNA of each sample to 1ng/ul in 30 µl of RNAse/DNAse free water. 

[GUIDELINES]
To minimize the risk of contamination, steps in this protocol must be performed in a DNAse-free environment, preferably in a disinfected biosafety cabinet. 
This protocol was found suitable to prepare a sequencing library for SARS-CoV-2 amplicons generated using the ARTIC method and implemented without using PCR plates during library prep. However, this protocol may be effective for other kinds of amplicon sequencing. Please follow the appropriate reagent storage and handling precautions defined by the manufacturer.  

[STEPS]
SECTION: Overview of the library prep workflow
1.   
Please make sure you review and understand this library preparation workflow. 
SECTION: DNA tagmentation
2. This step uses the Nextera Transposome to tagment genomic DNA. Tagmentation is a  process that fragments DNA and then tags the fragmented DNA with adapter sequences in a single reaction.

SECTION: DNA tagmentation
3. Vortex BLT vigorously for 10 s  seconds to resuspend. Do not centrifuge BLT. 

SECTION: DNA tagmentation
4. Prepare Tagmentation master mix by adding the following to a separate tube for the master mix.  
BLT:   11 µL 
TB1:  11 µL 
Multiply the volume of BLT and TB1 by the number of samples.
SECTION: DNA tagmentation
5. Vortex tagmentation master mix thoroughly to resuspend BLT in tagmentation buffer.
SECTION: DNA tagmentation
6. Distribute 20 µL  into each 0.2 tubes containing 30 µL  of DNA samples (1 ng/μl ) and gently pipette 10 times to resuspend. Gentle pipetting prevents liquid drops splits on the walls of tubes. Do not spin down. 

SECTION: DNA tagmentation
7. Close caps and place tubes into the pre-heated thermocycler and resume the "TAG" program.
SECTION: DNA tagmentation
8. Immediately proceed to the next step --> Post-Tagmentation cleanup.
SECTION: Post tagmentation cleanup
9. This step washes the adapter-tagged DNA on the BLT before PCR amplification.
SECTION: Post tagmentation cleanup
9.1. Prepare the following reagents: 
-Tagment Stop Buffer (TSB): if precipitates are observed, heat at 37°C for 10 min (using a heat-block) and vortex to dissolve the precipitate. 
-Tagment Wash Buffer (TWB)
-DynaMag magnetic stand
-0.2 ml PCR tubes 
SECTION: Post tagmentation cleanup
9.2. Run the PTC program below on the thermocycler and press "pause".
Choose the preheat lid option and set it to 100 °C 
Set the reaction volume to 60 μl
37 °C for 15 min min
Hold at 10 °C

SECTION: Post tagmentation cleanup
10.  Add  10 µL of TSB to the tagmentation reaction and slowly pipette each well 10 times to resuspend the beads.
SECTION: Post tagmentation cleanup
11. Place tubes in the pre-programmed thermal cycler, and run the "PTC" program.
SECTION: Post tagmentation cleanup
12. Transfer content of  each PCR tube (60 µL ) to a 1.5 ml low adhesion microcentrifuge tube.

 

SECTION: Post tagmentation cleanup
15. Washing steps:

SECTION: Post tagmentation cleanup
13. Place tubes on the magnetic stand and wait until liquid is clear (~3 minutes).
SECTION: Post tagmentation cleanup
14. Using a P100 pipette, carefully remove and discard supernatant.
SECTION: Amplify tagmented DNA
16. This step amplifies the tagmented DNA through a limited-cycle PCR program. The PCR step adds Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation. 
 

SECTION: Amplify tagmented DNA
17. Fill the table below including index information.
 
 
            N701      N702      N703      N704      N705      N706      N707      N708        S502                                                                S503                                                                S504                                                                S505                                                                S506                                                                S507                                                                S508                                                                S517                                                           

 
 
SECTION: Amplify tagmented DNA
18. Pre-heat the thermocycler by running the "BLT" program below and press "Pause".

Choose the preheat lid option and set it to 100°C. set thermocycler: 
68°C for 3 minutes
98°C for 3 minutes
(6) cycles of:98°C for 45 seconds, 62°C for 30 seconds, 68°C for 2 minutes
68°C for 1 minute
Hold at 10°C
SECTION: Amplify tagmented DNA
19. Label 0.2 ml PCR tubes with the sample number.
SECTION: Amplify tagmented DNA
20. Combine the following volumes  to prepare the PCR master mix. Multiply each volume by the number of  samples being processed. Reagent overage is included in the volume to ensure accurate pipetting
EPM : 22 µL  
Nuclease-free water 22 µL 


SECTION: Amplify tagmented DNA
21. Briefly vortex then  centrifuge for 10 seconds.
SECTION: Amplify tagmented DNA
22. With the 1.5 ml tubes on the magnetic stand, use a P300 pipette to carefully remove and discard the supernatant. 
Foam that remains on the well-walls does not adversely affect the library.
SECTION: Amplify tagmented DNA
23. Remove tubes from the magnetic stand.
SECTION: Amplify tagmented DNA
24. Immediately add 40 µL of PCR master mix directly onto the beads in each sample tube, then gently pipette to mix until the beads are fully re-suspended and transfer all the contents into labeled PCR tubes. Gentle pipetting avoids bubbles and liquid drop splits on the walls of the tube.

SECTION: Amplify tagmented DNA
25. Add the 5µl of I5 index adapters and  5µl of I7 index adapters to each sample, using 1 pair of gloves per index adapters to minimize contamination risk.
SECTION: Amplify tagmented DNA
26. After adding each index, mix by gently pipetting up and down 5 times. Careful pipetting ensures the absence of bubbles and liquid splits on the tube walls.

SECTION: Amplify tagmented DNA
27. Place on the thermal cycler and run the "BLT" PCR program.  
 

 

SECTION: Cleaning the library
28. This step uses a double-sided bead purification procedure to purify the amplified libraries. 
Items to prepare : 
Sample Purification Beads (SPB): bring at room temperature 30 min before use, vortex to resuspend the beads, and invert to mix.
Freshly prepared 80% ethanol (EtOH). use milli-Q water to prepare the 80% ethanol. 
RSB (Resuspension Buffer): thaw and bring to room temperature;  vortex to mix.
1.5 ml low binding microcentrifuge tubes; label two tubes per sample.
Nuclease-free water.


SECTION: Cleaning the library
29. Transfer the contents to a 1.5 ml tube.
SECTION: Cleaning the library
30. Spin down for 10 seconds to collect contents at the bottom of tube.
SECTION: Cleaning the library
31. Place the tubes on the magnetic stand and wait until the liquid is clear (~5 minutes).
SECTION: Cleaning the library
32. Transfer 45 µl supernatant from each tube to new 1.5 ml tubes.
SECTION: Cleaning the library
33. Vortex and invert SPB multiple times to resuspend.
SECTION: Cleaning the library
34. The starting DNA was a small PCR amplicon input. Perform the following steps:
SECTION: Cleaning the library
34.1. Add 81 µl SPB to each 1.5 ml tube containing supernatant. 
SECTION: Cleaning the library
34.2. Mix using a plate shaker at 1600 rpm for 1 minute.

SECTION: Cleaning the library
35. Incubate caped tubes at room temperature for 5 minutes.
SECTION: Cleaning the library
36. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
SECTION: Cleaning the library
37. Without disturbing the beads, remove and discard supernatant.
SECTION: Cleaning the library
38. Wash two times as follows:
SECTION: Cleaning the library
38.1. With the plate on the magnetic stand, add 200 µl fresh 80% EtOH without mixing.
SECTION: Cleaning the library
38.2. Incubate for 30 seconds.
SECTION: Cleaning the library
38.3. Without disturbing the beads, remove and discard supernatant.
SECTION: Cleaning the library
39. Use a 20 µl pipettor to remove and discard residual EtOH.
SECTION: Cleaning the library
40. Air-dry on the magnetic stand for 5 minutes, then remove from the magnetic stand.
SECTION: Cleaning the library
41. Add 32 µl RSB to the beads and gently pipette 5 times to resuspend.
SECTION: Cleaning the library
42. Incubate at room temperature for 2 minutes.
SECTION: Cleaning the library
43. Place the tube on the magnetic stand and wait until the liquid is clear (~2 minutes).
SECTION: Cleaning the library
44. Transfer 30 µl supernatant to a new low binding 0.5 ml tube. This supernatant contains double stranded DNA library.
 

SECTION: Pool and dilute library
45. Library quality control
Check the concentration of library using       

SECTION: Pool and dilute library
46. Run 1 µl of undiluted library on an Agilent Technology 2100 Bioanalyzer using the  . 


 
 
SECTION: Pool and dilute library
47. Dilute Libraries to the starting concentration     

SECTION: Pool and dilute library
48. Dilute the library using RSB to 2 Nanomolar (nM) (10 µL) into a low binding 0.6 ml  tubes. 

 


 

SECTION: Pool and dilute library
49. Library  pooling
Label a clean 1.5 ml low binding microcentrifuge tube.

SECTION: Pool and dilute library
49.1. 
Add 5ul of each 2nM diluted libraries into the 1.5 ml tube to make a pooled 2nM DNA library. 
	
SECTION: Pool and dilute library
49.2. Mix for 2 min 30 seconds at 1400 rpm. 
SECTION: Pool and dilute library
50. Library denaturation before the sequencing using Illumina MiSeq v2
Source: https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/miseq-denature-dilute-libraries-guide-15039740-10.pdf

Prepare :  , NaOH (10 M), milli-Q water (ultrapure water), 1.5 ml low binding tubes.

	• thaw HT1 ((Hybridization Buffer), RSB,   and place on ice.
	• prepare 1N NaOH = 10M NaOH (50ul) + MilliQ H2O (450ul)
	• prepare fresh [0.2N NaOH] from 1N NaOH stock solution : 
		○ Mix 800 µLmilli-Q water + 200 µLof Stock 1N NaOH
		○ Invert tube several times to mix
NB: 0.2N NaOH should be used within 12 hours. 
SECTION: Pool and dilute library
50.1. Label a new 1.5 ml low binding microcentrifuge tube and combine the following volumes in the tube:
- 5 μl  of pooled library (2nM) 
- 5 μl of 0.2N NaOH
SECTION: Pool and dilute library
50.2. Briefly vortex the mixture.
SECTION: Pool and dilute library
50.3. briefly centrifuge for 2- 3 seconds.
SECTION: Pool and dilute library
50.4. incubate for 5 minutes at room temperature.
SECTION: Pool and dilute library
50.5. Add 990 µL of pre-chilled HT1 into the tube to obtain 10 Picomolar (pM) of  denatured library (1 mL) --> then flick tube to mix.

SECTION: Pool and dilute library
50.6. Dilute denatured library (if needed)
  

SECTION: Pool and dilute library
50.7. Please make a 10 Picomolar (pM)of final solution (600 µL). Therefore, 
- transfer 600 µL of denatured library into new low binding  1.5 ml tube. 
- Invert to mix and spin down 
SECTION: Pool and dilute library
51. Preparation of PhiX control
PhiX Control v3 is a reliable, adapter-ligated library used as a control for Illumina sequencing runs. The library is derived from the small, well-characterized PhiX genome, offering several benefits for sequencing and alignment.
This is a small, ready-to-use Illumina library with a balanced nucleotide representation. Adding a 2% PhiX spike-in to your library provides additional metrics. For low-diversity libraries, use a 10% spike-in to increase base diversity.
SECTION: Pool and dilute library
51.1. In a 0.2 ml PCR tube, prepare 4 Nanomolar (nM) Phix from the 10 Nanomolar (nM) stock. 
Mix:
	•2 µL (of 10 nM stock) 
	• 3 µL of  RSB

SECTION: Pool and dilute library
51.2. In a clean1.5 ml tube, Mix : 
	• 4 nM PhiX library: 5 µL
	• 0.2 N NaOH: 5 µL

SECTION: Pool and dilute library
51.3. Vortex the modified PhiX solution vigorously.
SECTION: Pool and dilute library
51.4. Briefly centrifuge.
SECTION: Pool and dilute library
51.5. Incubate at room temperature for 5 minutes.
SECTION: Pool and dilute library
51.6. Dilute denatured PhiX to 20 Picomolar (pM)

SECTION: Pool and dilute library
51.7. Mix : 
	• Modified PhiX solution: 10 µL
	• Ice-cooled HT1: 990 µL
The newly prepared 1 mL of PhiX solution has a final concentration of 20 pM. It can be kept frozen for up to 3 weeks.
SECTION: Pool and dilute library
51.8. To prepare 10 pM of PhiX control, mix : 
	• 240 µL of  PhiX solution (20 pM),
	• 240 µL of ice cooling HT1.

SECTION: Pool and dilute library
51.9. In a new e-tube, prepare the PhiX 10% solution by mixing : 
	• PhiX (10 pM): 60 µL
	•  DNA library (10 pM): 540 µL

SECTION: Pool and dilute library
51.10. Store the prepared PhiX control and library pool at -30 °C until sequencing using MiSeq flowcell reagents.

SECTION: DNA tagmentation
2.1. Thaw EPM and indexes on ice.
SECTION: DNA tagmentation
2.2. Bring the following reagents at room temperature: BLT (beads linked transposomes), TB1(Tagmentation buffer), TSB (Tagment Stop buffer), and TWB (Tagment Wash Buffer).
SECTION: DNA tagmentation
2.3. Vortex BLT and TB1 to mix before use.
SECTION: DNA tagmentation
2.4. Pre-heat the lid of the Thermocycler (by Running the "TAG" program, and press "Pause")
The TAG program is set as follows: 
Set the reaction volume  to 50 µL 
55 °C  for 15 min 15 min
Hold at 10 °C 
SECTION: Post tagmentation cleanup
15.1. Remove from the magnetic stand and use a deliberately slow pipetting technique to add 100 µl TWB directly onto the beads. The deliberately slow pipetting technique minimizes the potential of TWB foaming to avoid incorrect volume aspiration and incomplete mixing.
SECTION: Post tagmentation cleanup
15.2. Mix at 1400 rpm for 2 min at room temperature (or pipette mix slowly 10 times until beads are re-suspended).
SECTION: Post tagmentation cleanup
15.3. Place the tubes on the magnetic stand and wait until the liquid is clear (~3 minutes).
SECTION: Post tagmentation cleanup
15.4. Using a P200 pipette, remove and discard the supernatant. 
SECTION: Post tagmentation cleanup
15.5. Repeat the washing steps 15.1 to 15.3  above. 
SECTION: Post tagmentation cleanup
15.6. Keep tubes on the magnetic stand until step 22 of the section Amplify Tagmented DNA section below. The TWB remains in the tube to prevent overdrying of the beads.