TITLE: Illumina Nextera DNA Flex library construction and sequencing for SARS-CoV-2: Adapting  COVID-19 ARTIC protocol
AUTHORS: Sureshnee Pillay, Jennifer Giandhari, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, Vagner Fonseca, Marta Giovanetti, Luiz Carols Alcantara, Tulio de Oliveira, Jennifer Giandhari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing amplicons tiling the viral genome sequencing. It uses the V3 nCov-2019 primers  from the ARTIC network. This is followed by library construction using Nextera Flex, which we found to save 9h of hands on time as compared with original protocol that use TrueSeq for library contstruction. It also describes the pooling of samples and quantitation, prior to sequencing on the Illumina Miseq and NextSeq. </div><div class = "text-block">It is adapted from the nCov-2019 sequencing protocol from Quick and colleagues, which can be found here: </div></div>

[STEPS]
?. [cDNA]
Prepare the cDNA mastermix in the pre-PCR clean room. The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
?. [cDNA]
Mix the following components in a labeled 1.5ml Component:  AB1ComponentVolume (ul)250μM Random Hexamers 11310mM dNTPs mix (10mM each) 114Template RNA115Total12
AB1ComponentVolume (ul)250μM Random Hexamers 11310mM dNTPs mix (10mM each) 114Template RNA115Total12
?. [cDNA]
Add  to a labeled 1.5ml eppendorf tube.
[50μM Random Hexamers 1]
?. [cDNA]
Add .
[10mM dNTPs mix (10mM each) 1]
?. [cDNA]
Add .
[Template RNA]
The total volume in the tube should now be  .
?. [cDNA]
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
?. [cDNA]
Aliquot the mastermix in labelled PCR strip tubes.
PCR master mixes (shown in Tables 1 and 3) can be prepared at the same time, in the pre-PCR area before starting amplifications.
?. [cDNA]
Incubate the reaction as follows in a thermal cycler.  AB1Temperature (°C)Time2655 minutes341 minute
AB1Temperature (°C)Time2655 minutes341 minute
?. [cDNA]
Spin down the tubes with the RNA and primers to get all liquid to the bottom.
?. [cDNA]
Prepare the following mastermix in the clean mastermix room.Mix the following components in a labeled 1.5ml eppendorf tube:  AB1ComponentVolume (μl)2SSIV Buffer43100mM DTT14RNaseOUT RNase Inhibitor15SSIV Reverse Transcriptase16Total7
AB1ComponentVolume (μl)2SSIV Buffer43100mM DTT14RNaseOUT RNase Inhibitor15SSIV Reverse Transcriptase16Total7
?. [cDNA]
Add  to a labeled 1.5ml eppendorf tube.
[SSIV Buffer]
?. [cDNA]
Add .
[RNaseOUT RNase Inhibitor]
?. [cDNA]
Add .
[100mM DTT]
?. [cDNA]
Add .
[SSIV Reverse Transcriptase]
The total volume should now be  .
?. [cDNA]
The mastermix must be added to the  for a .
[denatured RNA]
[total volume]
?. [cDNA]
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
?. [cDNA]
Incubate the reaction as follows in a thermal cycler.  AB1Temperature (°C)Time24250 minutes37010 minutes45Hold
AB1Temperature (°C)Time24250 minutes37010 minutes45Hold
?. [Primer Pool Preparation]
Primers must be diluted and pooled using nuclease free water in a clean mastermix hood. The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
?. [Primer Pool Preparation]
If required, resuspend lyophilised primers at a concentration of  each.
2019- nCoV primers for this protocol were designed using Primal Scheme to generate overlapping 400 nucleotide amplicons.
?. [Primer Pool Preparation]
To generate , add  (named pool 1 or pool 2) to a 1.5ml eppendorf tube labeled either “Pool 1 (100μM)” or “Pool 2 (100μM)”.
[primer pool stocks]
[of each primer pair]
Total volume will be   for Pool 1 (100uM) and   for Pool 2 (100uM). These are now   stocks of each primer.
?. [Primer Pool Preparation]
Dilute the 100μM primer pool 1:10 in molecular grade water, to generate .
[primer stocks]
It is recommended that multiple aliquots of each primer pool are made in case of degradation or contamination.
?. [Primer Pool Preparation]
Primers need to be used at a final concentration of  . In this case both pools have 98 primers in, so the requirement is   per  .
?. [Tiling PCR]
Prepare the PCR mastermix in the clean mastermix room.
?. [Tiling PCR]
The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
?. [Tiling PCR]
A mastermix for each pool must be made up in the mastermix hood.Mix the following components in a labeled 1.5ml eppendorf tube:  ABC1ComponentPool 1 volumes (μl)Pool 2 volumes (μl)25X Q5 Reaction Buffer55310mM dNTPs0.50.54Q5 Hot Start DNA Polymerase0.250.255Primer Pool 1 or 2 (10μM)3.63.66Nuclease-free water10.6510.657Total2020
ABC1ComponentPool 1 volumes (μl)Pool 2 volumes (μl)25X Q5 Reaction Buffer55310mM dNTPs0.50.54Q5 Hot Start DNA Polymerase0.250.255Primer Pool 1 or 2 (10μM)3.63.66Nuclease-free water10.6510.657Total2020
?. [Tiling PCR]
Add  to a labeled 1.5ml eppendorf tube.
[Table 5. PCR mastermix]
?. [Tiling PCR]
Add .
[10mM dNTPs]
?. [Tiling PCR]
Add .
[Q5 Hot Start DNA Polymerase]
?. [Tiling PCR]
Add .
[Primer Pool 1 or 2 (10μM)]
?. [Tiling PCR]
Add .
[Nuclease-free water]
The total volume should now be  .
?. [Tiling PCR]
Aliquot the mastermix in labelled PCR strip tubes.
?. [Tiling PCR]
Add  under the extraction hood or general lab hood, which has been decontaminated using with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
[of cDNA]
?. [Tiling PCR]
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
?. [Tiling PCR]
Incubate the reaction as follows in a thermal cycler.  ABCD1StepTemperature (°C)TimeCycles2Heat Activation9830 seconds13Denaturation9815 seconds354Annealing655 minutes5Hold4∞
ABCD1StepTemperature (°C)TimeCycles2Heat Activation9830 seconds13Denaturation9815 seconds354Annealing655 minutes5Hold4∞
?. [PCR Clean-up]
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into a single 1.5 ml eppendorf tube.
?. [PCR Clean-up]
Vortex Ampure beads thoroughly to ensure they are well resuspended; the solution should be a homogenous brown colour.
?. [PCR Clean-up]
Add an equal volume (1:1) of Ampure beads to the pooled sample tube and mix gently by either flicking or pipetting.
For example, add   to a  .
?. [PCR Clean-up]
Pulse centrifuge to collect all liquid at the bottom of the tube.
?. [PCR Clean-up]
Incubate for  at .
0 Room temperature
?. [PCR Clean-up]
Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. [PCR Clean-up]
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. [PCR Clean-up]
Add  (at ) to the pellet.
[of freshly prepared 70% ethanol]
0 Room temperature
?. [PCR Clean-up]
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
?. [PCR Clean-up]
Add  (at ) to the pellet.
[of freshly prepared 70% ethanol]
0 Room temperature
?. [PCR Clean-up]
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
?. [PCR Clean-up]
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
?. [PCR Clean-up]
With the tube lid open incubate for  or until the pellet loses its shine.
If the pellet dries completely it will crack and become difficult to resuspend
?. [PCR Clean-up]
Resuspend pellet in , mix gently by either flicking or pipetting and incubate for .
[Elution Buffer (EB)]
?. [PCR Clean-up]
Place on magnetic stand and transfer sample to a clean 1.5mL eppendorf tube ensuring no beads are transferred into this tube.
?. [PCR Clean-up]
***Sample concentration can be determined using the Qubit and the size of amplicons can be visualized using the LabChip Fragment Analyzer.
The expected amplicon size is 400bp.
?. [Library Preparation Tagment Amplicon DNA]
ABC1ItemStorageInstructions2BLT(bead-linked transposomes)2°C to 8°CBring to room temperature. Vortex to mix. Do not centrifuge before pipetting.3TB1(Tagmentation buffer)-25°C to -15°CBring to room temperature. Vortex to mix.
ABC1ItemStorageInstructions2BLT(bead-linked transposomes)2°C to 8°CBring to room temperature. Vortex to mix. Do not centrifuge before pipetting.3TB1(Tagmentation buffer)-25°C to -15°CBring to room temperature. Vortex to mix.
?. [Library Preparation Tagment Amplicon DNA]
Add – to each well of a 96-well PCR plate / 0.2ml strip tubes so that the total input amount is 100–500 ng.
2 µl
[DNA]
?. [Library Preparation Tagment Amplicon DNA]
If DNA volume
?. [Library Preparation Tagment Amplicon DNA]
Vortex BLT vigorously for  to resuspend.
?. [Library Preparation Tagment Amplicon DNA]
Vortex in between adding BLT as necessary.
?. [Library Preparation Tagment Amplicon DNA]
Prepare the tagmentation master mix.Multiply each volume by the number of samples being processed  AB1ComponentVolume (μl) per sample2BLT113TB1114Total22
AB1ComponentVolume (μl) per sample2BLT113TB1114Total22
?. [Library Preparation Tagment Amplicon DNA]
Please scale this step as needed. For :----
1
[BLT]
[TB1]
[Total]
?. [Library Preparation Tagment Amplicon DNA]
Vortex the tagmentation master mix thoroughly.
?. [Library Preparation Tagment Amplicon DNA]
Transfer  to each well of the plate containing a sample.
[tagmentation master mix]
Use fresh tips for each sample column.
?. [Library Preparation Tagment Amplicon DNA]
Resuspend by pipetting each sample 10 times.
?. [Library Preparation Tagment Amplicon DNA]
Seal the plate with a plate sealer, place on the preprogrammed thermal cycler, and run the tagmentation program.  AB1Temperature (°C)Time25515 minutes310Hold
AB1Temperature (°C)Time25515 minutes310Hold
?. [Post Tagmentation Clean-up]
ABC1ItemStorageInstructions2TSB (Tagment stop buffer)15°C to 30°CIf precipitates are observed, heat at 37°C for 10 minutes, and then vortex until precipitates are dissolved. Use at room temperature.3TWB (Tagment wash buffer)15°C to 30°CUse at room temperature.  AB1Temperature (°C)Time23715 minutes310Hold
ABC1ItemStorageInstructions2TSB (Tagment stop buffer)15°C to 30°CIf precipitates are observed, heat at 37°C for 10 minutes, and then vortex until precipitates are dissolved. Use at room temperature.3TWB (Tagment wash buffer)15°C to 30°CUse at room temperature.
AB1Temperature (°C)Time23715 minutes310Hold
?. [Post Tagmentation Clean-up]
Resuspend the beads by slowly pipetting each well/ tube 10 times.
?. [Post Tagmentation Clean-up]
Seal the plate with / tubes, place on the preprogrammed thermal cycler, and run the post tagmentation cleanup  program.
?. [Post Tagmentation Clean-up]
Place the plate on the magnetic stand for approximately  until liquid is clear.
?. [Post Tagmentation Clean-up]
Using a multichannel pipette, remove and discard supernatant.
?. [Post Tagmentation Clean-up]
Remove the sample plate from the magnetic stand and use a deliberately slow pipetting technique to add  directly onto the beads.
[TWB ]
This slow pipetting technique minimizes the potential of TWB foaming to avoid incorrect volume aspiration and incomplete mixing.
?. [Post Tagmentation Clean-up]
Slowly pipette until beads are fully resuspended.
?. [Post Tagmentation Clean-up]
Place the plate on the magnetic stand for approximately  until liquid is clear.
?. [Post Tagmentation Clean-up]
Using a multichannel pipette, remove and discard supernatant.
?. [Post Tagmentation Clean-up]
Remove the plate from the magnetic stand and use a deliberately slow pipetting technique to add  directly onto the beads.
[TWB]
?. [Post Tagmentation Clean-up]
Slowly pipette each well/tube to resuspend the beads.
?. [Post Tagmentation Clean-up]
Place the plate on the magnetic stand for approximately  until liquid is clear.
?. [Post Tagmentation Clean-up]
Using a multichannel pipette, remove and discard supernatant.
?. [Post Tagmentation Clean-up]
Remove the plate from the magnetic stand and use a deliberately slow pipetting technique to add  directly onto the beads.
[TWB]
?. [Post Tagmentation Clean-up]
Slowly pipette each well/tube to resuspend the beads.
?. [Post Tagmentation Clean-up]
Seal the plate and keep on the magnetic stand until step 69 of the Procedure section in Amplify Tagmented DNA.
?. [Post Tagmentation Clean-up]
The TWB remains in the wells to prevent overdrying of the beads.
?. [Post Tagmentation Clean-up]
Add  to the tagmentation reaction.
[TSB]
?. [Amplify Tagment DNA]
ABC1ItemStorageInstructions2EPM (enhanced PCR MIx)-25°C to -15°CThaw on ice. Invert to mix, then briefly centrifuge.3Index Adapters (Plates)-25°C to -15°CThaw at room temperature. Spin briefly before
use.Combine the following volumes to prepare the PCR master mix. Multiply each volume by the number of samples being processed.  AB1ComponentVolume (μl) per sample2EPM223NFW224Total44
ABC1ItemStorageInstructions2EPM (enhanced PCR MIx)-25°C to -15°CThaw on ice. Invert to mix, then briefly centrifuge.3Index Adapters (Plates)-25°C to -15°CThaw at room temperature. Spin briefly before
use.
AB1ComponentVolume (μl) per sample2EPM223NFW224Total44
Reagent overage is included in the volume to ensure accurate pipetting.
?. [Amplify Tagment DNA]
Please scale this step as needed. For :------
1
[EPM]
[NFW]
[Total ]
?. [Amplify Tagment DNA]
Vortex and centrifuge the PCR master mix at .
Centrifuge: 280 34, 10 s
?. [Amplify Tagment DNA]
With the plate on the magnetic stand, use a 200 μl multichannel pipette to remove and discard supernatant. (from step 65 of post tagmentation clean-up)
Foam that remains on the well walls does not adversely affect the library.
?. [Amplify Tagment DNA]
Remove from the magnet.
?. [Amplify Tagment DNA]
Immediately add  directly onto the beads in each sample well/ tube.
[PCR master mix]
?. [Amplify Tagment DNA]
Pipette mix until the beads are fully resuspended.
Alternatively, seal the plate and use a plate shaker at 1600 rpm for  .
?. [Amplify Tagment DNA]
Seal the sample plate and centrifuge at .
Centrifuge: 280 34, 3 s
?. [Amplify Tagment DNA]
Add  of the appropriate index adapters to each sample.
10 µl
?. [Amplify Tagment DNA]
Using a pipette set to 40 μl, pipette 10 times to mix.
Alternatively, seal the plate/ tubes and use a plate shaker at 1600 rpm for  .
?. [Amplify Tagment DNA]
Centrifuge at .
Centrifuge: 280 34, 30 s
?. [Amplify Tagment DNA]
Place on the thermal cycler and run the Enrichment PCR program.  ABC1Temperature (°C)Time2683 minutes3983 minutes49845 seconds8 cycles56230 seconds6682 minutes7681 minute810Hold
ABC1Temperature (°C)Time2683 minutes3983 minutes49845 seconds8 cycles56230 seconds6682 minutes7681 minute810Hold
?. [Amplify Tagment DNA]
SAFE STOPPING POINT If you are stopping, store at  to  for up to  (3 days).
2 °C
8 °C
?. [Clean-up Libraries]
ABC1ItemStorageInstructions2SPB (Sample Purification Beads)2°C to 8°CLet stand at room temperature for 30 minutes. Vortex and invert to mix.3RSB (Resuspension Buffer)-25°C to -15°CThaw and bring to room temperature. Vortex to mix.
ABC1ItemStorageInstructions2SPB (Sample Purification Beads)2°C to 8°CLet stand at room temperature for 30 minutes. Vortex and invert to mix.3RSB (Resuspension Buffer)-25°C to -15°CThaw and bring to room temperature. Vortex to mix.
?. [Clean-up Libraries]
Prepare fresh  from absolute ethanol.
[EtOH]
?. [Clean-up Libraries]
Centrifuge at  to bring all contents to the bottom.
Centrifuge: 280 34, 1 min
?. [Clean-up Libraries]
Place the plate/ tubes on a magnetic stand for approximately  until liquid is clear.
?. [Clean-up Libraries]
Transfer  supernatant from each well of the PCR plate/ tubes to the corresponding well of a new plate/ tubes.
45 µl
?. [Clean-up Libraries]
Vortex and invert SPB multiple times to resuspend.
?. [Clean-up Libraries]
Add  to each well/ tube.
[nuclease-free water]
?. [Clean-up Libraries]
Add  to each well/ tube.
[SPB]
?. [Clean-up Libraries]
Mix well by pipetting 10 times.
Alternatively, seal the plate and use a plate shaker at 1600 rpm for  .
?. [Clean-up Libraries]
Incubate at  for .
0 Room temperature
?. [Clean-up Libraries]
Place on the magnetic stand for approximately  until the liquid is clear.During incubation, thoroughly vortex the SPB (undiluted stock tube), and then add  to each well of a new plate/tubes.
15 µl
?. [Clean-up Libraries]
Transfer  supernatant from each well of the first plate/ tubes into the corresponding well of the second plate/ tubes (containing 15 μl undiluted SPB).
125 µl
?. [Clean-up Libraries]
Mix well by pipetting 10 times.
Alternatively, seal the plate and use a plate shaker at 1600 rpm for  .
?. [Clean-up Libraries]
Discard the first plate/ tubes.
?. [Clean-up Libraries]
Pipette each well 10 times to mix.
?. [Clean-up Libraries]
Incubate at  for .
0 Room temperature
?. [Clean-up Libraries]
Place on the magnetic stand for approximately  until the liquid is clear.
?. [Clean-up Libraries]
Without disturbing the beads, remove and discard supernatant.
?. [Clean-up Libraries]
Wash two times as follows:
?. [Clean-up Libraries]
Add  with the plate on the magnetic stand.
[freshly prepared 80% ethanol]
?. [Clean-up Libraries]
Incubate for .
?. [Clean-up Libraries]
Without disturbing the beads, remove and discard the supernatant.
?. [Clean-up Libraries]
Add  with the plate on the magnetic stand.
[freshly prepared 80% ethanol]
?. [Clean-up Libraries]
Without disturbing the beads, remove and discard the supernatant.
?. [Clean-up Libraries]
Use a 20 μl pipette to remove any residual ethanol.
?. [Clean-up Libraries]
Air-dry on the magnetic stand for .
?. [Clean-up Libraries]
Remove from the magnetic stand.
?. [Clean-up Libraries]
Add  to each well/ tube.
[RSB]
?. [Clean-up Libraries]
Resuspend by pipette mixing.
?. [Clean-up Libraries]
Incubate at  for .
0 Room temperature
?. [Clean-up Libraries]
Place the plate/ tubes on the magnetic stand for approximately .
?. [Clean-up Libraries]
Transfer  to a new 96-well PCR plate/ tubes.
[supernatant]
?. [Clean-up Libraries]
SAFE STOPPING POINT If you are stopping, seal the plate, and store at  to  for up to 30 days.
-25 °C
-15 °C
?. [Normalization of DNA]
Quantify the DNA as described using the Qubit and determine the fragment length using the LabChip.
?. [Normalization of DNA]
Using the Qubit concentrations and fragment length normalize the libraries to equimolar 4nM by diluting with RSB buffer.
?. [Normalization of DNA]
Calculate appropriate amount of diluent in 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
?. [Normalization of DNA]
Pipette mix 5 times.
?. [Normalization of DNA]
Use a multi-channel pipette to transfer  to an 8 strip-tube and spin briefly.
[of the diluted sample library]
?. [Normalization of DNA]
Pool the library samples from the 8-strip tubes to a labelled Pooled Amplicon Library (PAL) 2ml eppendorf tube.
?. [Normalization of DNA]
Proceed to library denaturation.
?. [Library Denaturation]
Remove the tube of HT1 (Hybridization Buffer) from the freezer (-15°C to -25°C) and set aside at  to thaw.
-15 Room temperature
?. [Library Denaturation]
When thawed, store at  to  until you are ready to dilute denatured libraries.
2 °C
8 °C
?. [Library Denaturation]
Prepare 500μl of 0.2 N NaOH by combining the following volumes in a 1.5ml microcentrifuge tube:  and .Refer to the formula below:1M=1N10N(x)=(0.2)(500)x =  +
[laboratory-grade water]
[Stock 1.0 N NaOH]
[NaOH]
[laboratory-grade water]
A fresh dilution of 0.2N NaOH is required for the denaturation process in preparing sample DNA and a PhiX control.
?. [Library Denaturation]
Invert the tube several times to mix.
?. [Library Denaturation]
Combine the following volumes of pooled sample DNA and freshly diluted 0.2 N NaOH in a micro-centrifuge tube, by adding  to .
[of 4nM sample DNA]
[of 4nM sample DNA]
?. [Library Denaturation]
Discard the remaining dilution of 0.2 N NaOH or set aside to prepare a PhiX control within the next .
?. [Library Denaturation]
Vortex briefly to mix the sample solution, and then centrifuge the sample solution to .
Centrifuge: 280 34, 1 min
?. [Library Denaturation]
Incubate for  at  to denature the DNA into single strands.
8 Room temperature
?. [Library Denaturation]
Add  to .
[of 4nM sample DNA]
[of pre-chilled HT1]
The result is a 20pM denatured library in 1 mM NaOH.
?. [Library Denaturation]
Place the denatured DNA  or at  until you are ready to proceed to the final dilution.
on ice
4 °C
?. [Dilution of Denatured Library]
Use the following instructions to dilute the 20pM DNA further to give 600μl of the desired input concentration.Dilute the denatured DNA to the desired concentration using the following example (if using 5% PhiX):  ABCD1Final Concentration20pM denatured DNA5% PhiXPre-chilled HT1212pM356.4ul3.6ul240ul
ABCD1Final Concentration20pM denatured DNA5% PhiXPre-chilled HT1212pM356.4ul3.6ul240ul
?. [Dilution of Denatured Library]
Invert several times to mix and then pulse centrifuge.
?. [Dilution of Denatured Library]
To dilute PhiX to 4nM concentration, combine the following volumes in a microcentrifuge tube:• • ,  with
[of 10nM PhiX library]
[of 10mM Tris-Cl]
[Tween 20]
If not prepared within the last 12 hours, prepare a fresh dilution of 0.2 N NaOH.
?. [Dilution of Denatured Library]
Combine the following volumes in a micro-centrifuge tube:• •
[of 4 nM PhiX library]
[of 0.2 N NaOH]
?. [Dilution of Denatured Library]
Vortex briefly to mix.
?. [Dilution of Denatured Library]
Centrifuge at .
Centrifuge: 280 34, 1 min
?. [Dilution of Denatured Library]
Incubate at  for .
8 Room temperature
?. [Dilution of Denatured Library]
Dilute denatured PhiX to 20pM by adding pre-chilled HT1 to the denatured PhiX library as follows: • •
[denatured PhiX library]
[pre-chilled HT1]
?. [Dilution of Denatured Library]
Invert to mix.
?. [Dilution of Denatured Library]
Combine Library and PhiX Control.
?. [Dilution of Denatured Library]
Mix this solution well and briefly centrifuge.
?. [Dilution of Denatured Library]
Keep  or at  until it is ready to be loaded onto the MiSeq reagent cartridge.
on ice
4 °C