TITLE: Evaluation of miniaturized Illumina DNA preparation protocols for SARS-CoV-2 whole genome sequencing protocol
AUTHORS: Sureshnee Pillay, James Emmanuel San, Derek Tshiabuila, Yeshnee Naidoo, Yusasha Pillay, Akhil Maharaj, Ugochukwu J. Anyaneji, Eduan Wilkinson, Houriiyah Tegally, Richard J. Lessells, Cheryl Baxter, Tulio de Oliveira, Jennifer Giandhari

[DESCRIPTION]
This protocol describes the miniaturisation of the Illumina DNA Prep library kit and the use of a rapid modified protocol with fewer clean-up steps and different reagent volumes (full volume, half volume, one-tenth volume). The cost of sequencing can be reduced roughly sevenfold, and the time required to prepare the library can be reduced from 6.5 hours to 3 hours. The protocol adaptation represents a lower-cost, streamlined approach for SARS-CoV-2 sequencing, which can be used to generate genomic data quickly and affordably, particularly in resource-constrained settings.

[GUIDELINES]
Introduction 


This protocol describes a method for whole genome sequencing of the SARS-CoV-2 using a tiling PCR approach with overlapping primers and Nextera Flex libraries for Illumina sequencers. This method was produced in KRISP labs for the Network of Genomics Surveillance of South Africa (NGS-SA). 


Briefly, primers are designed to be 20-30bp in length and to generate 400bp amplicons with a 70bp overlap. The     primers are designed using an online tool called Primal Scheme (http://primal.zibraproject.org/).The amplicons generated can be sequenced on the on the Illumina MiSeq. This will produce next generation sequences covering the whole genome of the SARS-CoV-2 .


Purpose 


The purpose of this document is to provide detailed instructions that should be followed when performing the sequencing of SARS-CoV-2  whole genomes from RNA samples using the Nextera DNA Flex Library Kit.

[STEPS]
SECTION: cDNA
1. 
Prepare the cDNA master mix in the pre-PCR clean room. The master mix hood must be decontaminated before and after use with 10% extran and 70% ethanol, and sterilised with ultraviolet light (UV).
SECTION: cDNA
2. 
Mix the following components in a labeled 1.5ml tube:

 
     Component      Volume (ul)        50μM Random   Hexamers 1      1        10mM dNTPs mix   (10mM each) 1      1        Template RNA      11        Total      12   
 
SECTION: cDNA
2.1. 
Add 1 µl of LunaScript reagent into each well of a 96-well nested plate.
SECTION: cDNA
3. 
Incubate the reaction as follows in a thermal cycler.
 
 
ABTemperature (°C)Time25 °C2 minutes55 °C10 minutes95 °C1 minute4Hold
 
 
SECTION: cDNA
2.2. 
Transfer 5,5 ul of total nucleic acid extracts to each well as per Table 1.
SECTION: cDNA
2.3. 
The total volume in the tube should now be 6.5 µl.

SECTION: Primer Pool Preparation
4. 
Primers must be diluted and pooled in a clean master mix hood using nuclease-free water. The master mix hood must be decontaminated with 10% extran and 70% ethanol before and after use, and sterilised with ultraviolet light (UV).
SECTION: Primer Pool Preparation
5.                                                                                                                                                                                                                                                                                                                                                                                                                              To generate overlapping 400 nucleotide amplicons for this protocol, 2019-nCoV primers were designed using the Primal Scheme.
SECTION: Primer Pool Preparation
6. 
Resuspend lyophilised primers at a concentration of 100 M each if necessary.
SECTION: Primer Pool Preparation
7. 
To generate 100 µM primer pool stocks, add 5 µl of each primer pair (named pool 1 or pool 2) to a 1.5ml eppendorf tube labeled either “Pool 1 (100μM)” or “Pool 2 (100μM)”. Total volume will be 490 µl for Pool 1 (100uM) and 490 µl for Pool 2 (100uM). These are now 100 µM stocks of each primer.
SECTION: Primer Pool Preparation
8. To make 10M primer stocks, dilute the 100M primer pool 1:10 in molecular-grade water.
It is advised to make multiple aliquots of each primer pool in case of degradation or contamination.

SECTION: Tiling PCR
9. 
Prepare the PCR mastermix in the clean mastermix room.

SECTION: Tiling PCR
10. 
The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).

SECTION: Tiling PCR
11. 
A master mix for each pool must be made up in the master mix hood.
Mix the following components in a labelled 1.5ml eppendorf tube:
 
 
ComponentPool 1 volumes (μl)Pool 2 volumes (μl)5X Q5 Reaction Buffer2.52.510mM dNTPs0.250.25Q5 Hot Start DNA Polymerase0.1250.125Primer Pool 1 or 2 (10μM)1.81.8Nuclease-free water5.45.4Total1010
 
 
11.1. Make a master mix by multiplying each component by the number of samples being tested.
Add 2.5 µl   5X Q5 Reaction Buffer  to a labeled 1.5ml eppendorf tube. 


11.2. 
Add 0.25 µl 10mM dNTPs.
11.3. 
Add 0.125 µl Q5 Hot Start DNA Polymerase. 

11.4. 
Add 1.8 µl Primer Pool 1 or 2 (10μM). 
11.5. 
Add 5.4 µl Nuclease-free water.


12. 
Aliquot 10 µl  mastermix in labelled PCR strip tubes.

13. 
Add 2.5 µl of cDNA under the extraction hood or general lab hood, which has been decontaminated using 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
14. 
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
15. 
Incubate the reaction as follows in a thermal cycler.
 
     Step      Temperature (°C)      Time      Cycles        Heat Activation      98      30 seconds      1        Denaturation      98      15 seconds      35        Annealing      65      5 minutes        Hold      4      ∞     
 *Cycle number should be 25 for Ct 18-21, and up to a maximum of 35 cycles for Ct 35.
16. 
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into a single tube.
SECTION: Library Preparation: Tagmentation
17. 
Add 5 µl undiluted DNA to each well of a 96-well PCR plate / 0.2ml strip tubes.
SECTION: Library Preparation: Tagmentation
19. 
Vortex BLT vigorously for 00:00:10 to resuspend.
SECTION: Library Preparation: Tagmentation
20. 
Vortex in between adding BLT as necessary.
SECTION: Library Preparation: Tagmentation
21. 
Prepare the tagmentation master mix.

 
     Component      Volume (μl) per sample        BLT      11        TB1      11        Total      22   

SECTION: Library Preparation: Tagmentation
22. 
Vortex the tagmentation master mix thoroughly.
Transfer 2 µl tagmentation master mix to each well of the plate containing sample.
Use fresh tips for each sample column.
SECTION: Library Preparation: Tagmentation
21.1. 
 Please scale this step as needed. 

For 1 sample:

1 µl BLT
1 µl TB1
----
2 µl Total
SECTION: Library Preparation: Tagmentation
23. 
Resuspend by pipetting each sample 10 times.
SECTION: Library Preparation: Tagmentation
24. 
Seal the plate with a plate sealer, place on the preprogrammed thermal cycler, and run the tagmentation program.
 
     Temperature (°C)      Time        55      15 minutes        10      Hold   
 
SECTION: Library Preparation: Tagmentation
18. 
 
     BLT(bead-linked   transposomes)      2°C to 8°C      Bring to room   temperature. Vortex to mix. Do not centrifuge before pipetting.        TB1(Tagmentation   buffer)      -25°C to -15°C      Bring to room   temperature. Vortex to mix.   
 
SECTION: Stop Tagmentation
26. Add 1 µl TSB to the tagmentation reaction.


SECTION: Stop Tagmentation
25.  
     Item      Storage       Instructions        TSB (Tagment stop   buffer)      15 oC to 30 oC       If   precipitates are observed, heat at 37°C for 10 minutes, and then vortex until   precipitates are dissolved. Use at room temperature.        TWB (Tagment wash   buffer)      15 2oC to 30 oC      Use at room   temperature.   
 
SECTION: Stop Tagmentation
27. Resuspend the beads by slowly pipetting each well/ tube 10 times.

SECTION: Stop Tagmentation
28. Seal the plate with / tubes, place it on the preprogrammed thermal cycler, and run the stop tag programme.
SECTION: Post Tagmentation Clean
30. Place the plate on the magnetic stand for approximately 00:01:00 until liquid is clear.

SECTION: Post Tagmentation Clean
31. Using a multichannel pipette, remove and discard supernatant.

SECTION: Post Tagmentation Clean
32. Remove the sample plate from the magnetic stand and add 10 µl  TWB directly onto the beads using a deliberately slow pipetting technique.
To avoid incorrect volume aspiration and incomplete mixing, this slow pipetting technique reduces the possibility of TWB foaming.
SECTION: Post Tagmentation Clean
33. Slowly pipette until beads are fully resuspended.

SECTION: Post Tagmentation Clean
34. Place the plate on the magnetic stand for approximately 00:01:00 until liquid is clear.

SECTION: Post Tagmentation Clean
35. Using a multichannel pipette, remove and discard supernatant.

SECTION: Post Tagmentation Clean
36. Remove the plate from the magnetic stand and add 10 µl  TWB directly onto the beads using a deliberately slow pipetting technique.
SECTION: Post Tagmentation Clean
37. Slowly pipette each well/tube to resuspend the beads.

SECTION: Post Tagmentation Clean
38. Place the plate on the magnetic stand for approximately 00:01:00 until liquid is clear.

SECTION: Post Tagmentation Clean
39. Using a multichannel pipette, remove and discard supernatant.

SECTION: Post Tagmentation Clean
40. Remove the plate from the magnetic stand and use a deliberately slow pipetting technique to add 
10 µl TWB directly onto the beads.

SECTION: Post Tagmentation Clean
41. Slowly pipette each well/tube to resuspend the beads.

SECTION: Post Tagmentation Clean
42. Seal the plate and keep on the magnetic stand until step 47 of the Procedure section in Amplify Tagmented DNA.

SECTION: Post Tagmentation Clean
43. The TWB remains in the wells to prevent overdrying of the beads.

SECTION: Amplify Tagment DNA
44.  
     Item      Storage       Instructions        EPM (Enhanced PCR Mix)      -25°C to-15°C       Thaw on ice.   Invert to mix, then briefly centrifuge.        Index Adapters  Tubes/plates       -25°C to-15°C      Thaw at room temperature.    [Tubes] Vortex to mix, then centrifuge briefly. [Plates] Spin briefly before   use.   
                                                                                                                                                                                                                                                                     
SECTION: Amplify Tagment DNA
45.  
     Component      Volume (μl)   per sample        EPM      22        NFW (Nuclease Free Water)      22        Total      44   
 The following volumes were combined to prepare the PCR master mix (Table 13). Each volume was multiplied by the number of samples being processed. Reagent overage was included in the volume to ensure accurate pipetting. 
SECTION: Amplify Tagment DNA
46. Vortex and centrifuge the PCR master mix at 280 x g 00:00:10.
SECTION: Amplify Tagment DNA
47. With the plate on the magnetic stand, use a multichannel pipette to remove and discard the supernatant. (from step 42 of post-tagmentation clean-up).The foam that remains on the walls of the well does not adversely affect the library.
SECTION: Amplify Tagment DNA
48. Remove from the magnet.
SECTION: Amplify Tagment DNA
49. Immediately add 4 µl PCR master mix directly onto the beads in each sample well/ tube.
SECTION: Amplify Tagment DNA
50. Pipette mix until the beads are fully resuspended. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00.
SECTION: Amplify Tagment DNA
51. Seal the sample plate and centrifuge at 280 x g 00:00:30.
SECTION: Amplify Tagment DNA
52. Add 1 µl of the appropriate index adapters to each sample.
SECTION: Amplify Tagment DNA
53. Using a pipette set to 4 μl, pipette 10 times to mix. Alternatively, seal the plate/ tubes and use a plate shaker at 1600 rpm for 00:01:00.
SECTION: Amplify Tagment DNA
54. Centrifuge at 280 x g 00:00:30.
SECTION: Amplify Tagment DNA
55. Place on the thermal cycler and run the Enrichment PCR program.
SECTION: Amplify Tagment DNA
56.  
Temperature (°C)Time68°C3 minutes98°C3 minutes98°C45 seconds8 cycles62°C30 seconds68°C2 minutes68°C1 minute10°CHold
 
SECTION: Amplify Tagment DNA
57. SAFE STOPPING POINT 
If you are stopping, store at 2 °C to 8 °C for up to 72:00:00 (3 days).


SECTION: Clean-up Libraries
58.  
     Item      Storage       Instructions        SPB (Sample Purification Beads)      2°C to 8°C      Let stand at   room temperature for 30 minutes. Vortex and invert to mix.        RSB (Resuspension Buffer)      -25°C to-15°C      Thaw and   bring to room temperature. Vortex to mix.   
 
SECTION: Clean-up Libraries
59. Prepare fresh 80 % EtOH from absolute ethanol.
SECTION: Clean-up Libraries
60. Centrifuge at 280 x g 00:01:00 to bring all contents to the bottom.
SECTION: Clean-up Libraries
61. Place the plate/ tubes on a magnetic stand for approximately 00:01:00 until liquid is clear.
SECTION: Clean-up Libraries
62. Transfer 4.5 µl supernatant from each well of the PCR plate/ tubes to the corresponding well of a new plate/ tubes.
SECTION: Clean-up Libraries
63.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Add 4 µl nuclease-free water to each well/ tube.                                                                                                                                                                                               
SECTION: Clean-up Libraries
64. Vortex and invert SPB multiple times to resuspend                                                                    
SECTION: Clean-up Libraries
65. Add 4.5 µl SPB to each well/ tube.
SECTION: Clean-up Libraries
66. Mix well by pipetting 10 times. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00.
SECTION: Clean-up Libraries
67. Incubate at Room temperature for 00:01:00.
SECTION: Clean-up Libraries
68. Place on the magnetic stand for approximately 00:01:00 until the liquid is clear. During incubation, thoroughly vortex the SPB (undiluted stock tube), and then add 1.5 µl to each well of a new plate/tubes.
SECTION: Clean-up Libraries
69. Transfer 12.5 µl supernatant from each well of the first plate/ tubes into the corresponding well of the second plate/ tubes (containing 1.5 μl undiluted SPB).
SECTION: Clean-up Libraries
70. Mix well by pipetting 10 times. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00.
SECTION: Clean-up Libraries
71. Discard the first plate/ tubes.
SECTION: Clean-up Libraries
72. Pipette each well 10 times to mix.
SECTION: Clean-up Libraries
73. Incubate at Room temperature for 00:01:00.
SECTION: Clean-up Libraries
74. Place on the magnetic stand for approximately 00:01:00 until the liquid is clear.
SECTION: Clean-up Libraries
75. Without disturbing the beads, remove and discard supernatant.
SECTION: Clean-up Libraries
76. Wash two times as follows:
SECTION: Clean-up Libraries
77. Add 20 µl freshly prepared 80% ethanol with the plate on the magnetic stand.
SECTION: Clean-up Libraries
78. Incubate for 00:00:30.
SECTION: Clean-up Libraries
79. Without disturbing the beads, remove and discard the supernatant.
SECTION: Clean-up Libraries
80. Add 20 µl freshly prepared 80% ethanol with the plate on the magnetic stand.
SECTION: Clean-up Libraries
81. Without disturbing the beads, remove and discard the supernatant.
SECTION: Clean-up Libraries
82. Use a 20 μl pipette to remove any residual ethanol.
SECTION: Clean-up Libraries
83. Air-dry on the magnetic stand for 00:01:00.
SECTION: Clean-up Libraries
84. Remove from the magnetic stand.
SECTION: Clean-up Libraries
85. Add 13.5 µl RSB to each well/ tube.
SECTION: Clean-up Libraries
86. Resuspend by pipette mixing.
SECTION: Clean-up Libraries
87. Incubate at Room temperature for 00:01:00.
SECTION: Clean-up Libraries
88. Place the plate/ tubes on the magnetic stand for approximately 00:01:00.
SECTION: Clean-up Libraries
89. Transfer 12 µl supernatant to a new 96-well PCR plate/ tubes.
SECTION: Clean-up Libraries
91. SAFE STOPPING POINT 
If you are stopping, seal the plate, and store at -25 °C to -15 °C for up to 30 days.

SECTION: Normalization of DNA
92. Using the Qubit concentrations and fragment length normalize the libraries to equimolar 4nM by diluting with RSB buffer.
SECTION: Normalization of DNA
93. Calculate theappropriate amount of diluent on an excel sheet to add to respective sample libraries in order to achieve a 4nm library concentration, using the following formula: 
Nanomolar concentration = (ng/μl /660 x 500) x 10^6
SECTION: Normalization of DNA
94. Pipette mix 5 times.
SECTION: Normalization of DNA
95. Use a multi-channel pipette to transfer 5 µl of the diluted sample library to an 8 strip-tube and spin briefly.
SECTION: Normalization of DNA
96. Pool the library samples from the 8-strip tubes to a labelled Pooled Amplicon Library (PAL) 2ml eppendorf tube.
SECTION: Normalization of DNA
97. Proceed to library denaturation.
SECTION: Library Denaturation
98. Remove the tube of HT1 (Hybridization Buffer) from the freezer (-15°C to -25°C) and set aside at Room temperature to thaw.
SECTION: Library Denaturation
99. When thawed, store at 2 °C to 8 °C until you are ready to dilute denatured libraries.  

SECTION: Library Denaturation
100. Prepare 500μl of 0.2 N NaOH by combining the following volumes in a 1.5ml microcentrifuge tube: 490 µl laboratory-grade water and 10 µl Stock 1.0 N NaOH.  
Refer to the formula below:    1M=1N1M=1N 10N(x)=(0.2)(500)10N(x)=(0.2)(500)    x = 10 µl NaOH + 490 µl laboratory-grade water.
 A fresh dilution of 0.2N NaOH is required for the denaturation process in preparing sample DNA and a PhiX control.
SECTION: Library Denaturation
101. Invert the tube several times to mix.
SECTION: Library Denaturation
102. Combine the following volumes of pooled sample DNA and freshly diluted 0.2 N NaOH in a micro-centrifuge tube, by adding 5 µl of 4nM sample DNA to 5 µl of 4nM sample DNA.
SECTION: Library Denaturation
103. Discard the remaining dilution of 0.2 N NaOH or set aside to prepare a PhiX control within the next 12:00:00.
SECTION: Library Denaturation
104. Vortex briefly to mix the sample solution, and then centrifuge the sample solution to 280 x g 00:01:00.
SECTION: Library Denaturation
105. Incubate for 00:05:00 at Room temperature to denature the DNA into single strands.
SECTION: Library Denaturation
106. Add 990 µl of pre-chilled HT1 to the 10 µl of 4nM denatured library.                                                                                                                           The result is a 20pM denatured library in 1 mM NaOH.
SECTION: Library Denaturation
107. Place the denatured DNA On ice or at 4 °C until you are ready to proceed to the final dilution.
SECTION: Dilution of Denatured Library
108. Use the following instructions to dilute the 20pM DNA further to give 600μl of the desired input concentration.

SECTION: Dilution of Denatured Library
109. Dilute the denatured DNA to the desired concentration using the following example (if using 1% PhiX):

 
Final Concentration20pM denatured DNA1% PhiXPre-chilledHT112 pM354.6μl3.6μl240μl
 *This was found to be the optimal loading concentration when using a Miseq V2 500 cycle kit
SECTION: Dilution of Denatured Library
110. Invert several times to mix and then pulse centrifuge.
SECTION: Dilution of Denatured Library
111. To dilute PhiX to 4nM concentration, combine the following volumes in a microcentrifuge tube: • 2 µl of 10nM PhiX library • 3 µl of 10mM Tris-Cl, pH8.5 with 0.1 % Tween 20 If not prepared within the last 12 hours, prepare a fresh dilution of 0.2 N NaOH.
SECTION: Dilution of Denatured Library
112. Combine the following volumes in a micro-centrifuge tube: • 5 µl of 4 nM PhiX library • 5 µl of 0.2 N NaOH
SECTION: Dilution of Denatured Library
113. Vortex briefly to mix.
SECTION: Dilution of Denatured Library
114. Centrifuge at 280 x g 00:01:00.
SECTION: Dilution of Denatured Library
115. Incubate at Room temperature for 00:05:00.
SECTION: Dilution of Denatured Library
116. Dilute denatured PhiX to 20pM by adding pre-chilled HT1 to the denatured PhiX library as follows: 
990 µl pre-chilled HT1 to 10 µl denatured PhiX library •                                                                                                             
SECTION: Dilution of Denatured Library
117. Invert to mix.
SECTION: Dilution of Denatured Library
118. Combine Library and PhiX Control.
SECTION: Dilution of Denatured Library
119. Mix this solution well and briefly centrifuge.
SECTION: Dilution of Denatured Library
120. Keep On ice or at 4 °C until it is ready to be loaded onto the MiSeq reagent cartridge.
SECTION: Clean-up Libraries
90. Rapid Method:

Using an example of 96 samples:

Pool 4.5ul of each sample  after the enrichment step into a 1.5 µl tube= 432 µl (4.5 x 96).

Add 384 µl (4x 96) of nuclease free water.

Add 432 µl (4.5 X96) of SPB.

Vortex and incubate for 5 minutes.

Place on amagnetic rack for 3 minutes.

Transfer the supernatant to a clean 1.5 µl tube.

Add 144 µl (1.5 x 96) of SPB.

Vortex and incubate for 5 minutes.

Place on a magnetic rack for 3 minutes.

Wash twice with 1 ml of 80% ethanol.

Remove all traces of ethanol after the last wash.

Elute in 35 µl of resuspension buffer.
SECTION: Stop Tagmentation
29.  
 Temperature (°C)Time3715 minutes10Hold