TITLE: Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater
AUTHORS: Xuan Lin, Melissa  Glier, Kevin Kuchinski, Tenysha Ross-Van Mierlo, David McVea, John Tyson, Natalie Prystajecky, Ryan Ziels

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this work, we aim to access the performance of three different multiplex primer schemes, i.e. Swift amplicon SARS-CoV-2 panel (150bp amplicons), ARTIC V3 panel (400bp amplicons), and SARS-CoV-2 midnight panel (1200bp amplicons), for metatranscriptomic sequencing of SARS-CoV-2 for influent wastewater and primary sludge.</div><br/><div class = "text-block">This protocol is adapted from the Swift amplicon™ SARS-COV-2 protocol (150bp amplicon),  ARTIC V3 protocol (400bp amplicon), and "midnight" protocol (1200bp amplicon).</div><br/><div class = "text-block">Sequencing libraries are prepared with 1) Oxford Nanopore Ligation Sequencing Kit (SQK-LSK109) with Native Barcoding kit (EXP-NEB104 and EXP-NEB114), or 2) NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® with NEBNext® Multiplex Oligos for Illumina®.</div><br/><div class = "text-block">Links to the protocols are:</div><div class = "text-block"><span>Swift amplicon protocol (150bp): </span><a href="https://swiftbiosci.com/swift-amplicon-sars-cov-2-panel" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://swiftbiosci.com/swift-amplicon-sars-cov-2-panel/</span></a></div><div class = "text-block"><span>ARTIC V3 protocol (400bp): </span><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye?version_warning=no" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye?version_warning=no</span></a></div><div class = "text-block"><span>"midnight" protocol V4 (1200bp): </span><a href="https://dx.doi.org/10.17504/protocols.io.bh7hj9j6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.bh7hj9j6</span></a></div></div>

[STEPS]
?. [First strand cDNA synthesis]
Preparation of master mix
?. [First strand cDNA synthesis]
Prepare the following components in a 1.5 mL Eppendorf DNA LoBind tube for the number of samples that will be tested and dispense  to 0.2mL each PCR tubes. Briefly spin down the 0.2 mL tubes containing Master Mix RT_1 and keep ComponentVolume50µM random hexamers2 μL10mM dNTPs mix (10mM each)2 μL
[Master Mix RT_1]
on ice
ComponentVolume50µM random hexamers2 μL10mM dNTPs mix (10mM each)2 μL
?. [First strand cDNA synthesis]
This step should be conducted in the pre-PCR area (e.g. cleaned DNA hood). Keep all the Master Mix while doing this step.
on ice
?. [First strand cDNA synthesis]
Prepare the following components in a 1.5mL tube and keep the Master Mix RT_2  . ComponentVolume (μL)100 mM DTT2Ribonuclease Inhibitor (40 U/µL)25x SSIV Buffer8SSIV Reverse transcriptase2Nuclease free H2O10
on ice
ComponentVolume (μL)100 mM DTT2Ribonuclease Inhibitor (40 U/µL)25x SSIV Buffer8SSIV Reverse transcriptase2Nuclease free H2O10
?. [First strand cDNA synthesis]
Add  to each 0.2 mL tube containing the Master Mix RT_1
[RNA extract]
?. [First strand cDNA synthesis]
Mix by pipetting or flicking the tube, spin down briefly.
?. [First strand cDNA synthesis]
Incubate the reaction mix in a thermocycler at  for  , then spin down briefly, and incubate immediately  for at least  .
65 °C
on ice
?. [First strand cDNA synthesis]
While on ice, add to each tube ,
[Master Mix RT_2]
?. [First strand cDNA synthesis]
Gently mix by pipetting or flicking the tubes and briefly spin down.
?. [First strand cDNA synthesis]
Incubate the reaction mix in the thermocycler for:  Step    Temp    Time    1    42 °C    00:50:00     2    70 °C    00:10:00        3    4°C    hold
Step    Temp    Time    1    42 °C    00:50:00     2    70 °C    00:10:00        3    4°C    hold
The cDNA can be stored at   . If needed, it is a safe stop point. The remaining RNA should be stored at   .
?. [cDNA cleanup and concentration]
cDNA cleanup with (all centrifuge steps at 13,000xg)
?. [cDNA cleanup and concentration]
Add   and  to the  .
[0.5M EDTA]
[1N NaOH]
[RT reaction]
?. [cDNA cleanup and concentration]
Incubate the reaction mix in a thermocycler at  for   to hydrolyze RNA.
65 °C
?. [cDNA cleanup and concentration]
Transfer the hydrolysis reaction mix from the last step to a new 1.5 mL Eppendorf DNA LoBind tube and add  to .  Briefly vortex to mix, and pulse spin to collect thesample.
[(7x volume)]
[hydrolysis reaction mix]
?. [cDNA cleanup and concentration]
Transfer mixture to a provided Zymo-Spin™ Column in a Collection Tube.
?. [cDNA cleanup and concentration]
Centrifuge for  and discard the flow-through.
?. [cDNA cleanup and concentration]
Add to the column. Centrifuge for  .
200 µl
?. [cDNA cleanup and concentration]
Repeat step 3.6.
?. [cDNA cleanup and concentration]
Add to the column matrix and incubate at   for  .Primer SchemeElute volume (μL)Swift_150bp14ARTIC V3_400bp18.5Midnight_1200bp25
0 Room temperature
Primer SchemeElute volume (μL)Swift_150bp14ARTIC V3_400bp18.5Midnight_1200bp25
?. [cDNA cleanup and concentration]
Transfer the column to a 1.5 ml microcentrifuge tube, then centrifuge for   to elute the DNA and keep on ice.
?. [Multiplex PCR]
Prepare the following PCR mastermixs and keep  .
on ice
?. [Multiplex PCR]
Prepare PCR mastermix for Swift_150bp primer scheme.ComponentReaction 1Reaction 2Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µLSwift Pool 1 (15nM each primer)6.25 μL0Swift Pool 2 (15nM each primer)06.25 μLTotal18.75 μL18.75 μL
ComponentReaction 1Reaction 2Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µLSwift Pool 1 (15nM each primer)6.25 μL0Swift Pool 2 (15nM each primer)06.25 μLTotal18.75 μL18.75 μL
?. [Multiplex PCR]
Prepare PCR mastermix for ARTIC V3_400bp primer scheme.ComponentReaction 1Reaction 2Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µLV3 Pool 1 (15nM each primer)4 μL0V3 Pool 2 (15nM each primer)04 μLTotal16.5 μL16.5 μL
ComponentReaction 1Reaction 2Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µLV3 Pool 1 (15nM each primer)4 μL0V3 Pool 2 (15nM each primer)04 μLTotal16.5 μL16.5 μL
?. [Multiplex PCR]
Prepare PCR mastermix for midnight_1200bp primer scheme.ComponentReaction 1Reaction 2Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µLmidnight Pool 1 (15nM each primer)1.1 μL0midnight Pool 2 (15nM each primer)01.1 μLTotal13.6 μL13.6 μL
ComponentReaction 1Reaction 2Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µLmidnight Pool 1 (15nM each primer)1.1 μL0midnight Pool 2 (15nM each primer)01.1 μLTotal13.6 μL13.6 μL
?. [Multiplex PCR]
Add the corresponding amount of cDNA to each of the PCR reactions, mix by pipetting or flicking the tube and spin down.Primer SchemeVolume per reaction (μL)Swift_150bp6.25ARTIC V3_400bp8.5Midnight_1200bp11.4
Primer SchemeVolume per reaction (μL)Swift_150bp6.25ARTIC V3_400bp8.5Midnight_1200bp11.4
?. [Multiplex PCR]
Run in a thermal cycler using the following program:Swift_150bp primer schemeStepTemperatureTimeCyclesInitial denaturation  98 °C30 s1Denaturation  Annealing/Extension  98°C                   65°C15 s                    2 min35Hold4 °C ∞1ORARTIC V3 and midnight primer schemeStepTemperatureTimeCyclesInitial denaturation  98 °C30 s1Denaturation  Annealing/Extension  98°C                   65°C15 s                    5 min35Hold4 °C ∞1
StepTemperatureTimeCyclesInitial denaturation  98 °C30 s1Denaturation  Annealing/Extension  98°C                   65°C15 s                    2 min35Hold4 °C ∞1
StepTemperatureTimeCyclesInitial denaturation  98 °C30 s1Denaturation  Annealing/Extension  98°C                   65°C15 s                    5 min35Hold4 °C ∞1
For ARTIC V3 400bp primer scheme and midnight 1200bp primer scheme (sequencing with nanopore),  continue with steps 7-16.  For Swift 150bp primer scheme (sequencing with Illumina), continue with steps 17-26.
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Combine the  PCR reaction mixtrues for the two pools per sample into new 1.5 mL Eppendorf tubes, one per sample.
25 µl
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Add  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down.Incubate at  for  .
50 µl
0 Room temperature
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Prepare fresh 80% ethanol in nuclease-free water that enough for  per sample.
400 µl
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Keep the tubes on the magnet and wash the beads with   of freshly prepared 80% ethanol without disturbing the pellet. Incubate for  and pipette off the ethanol.
200 µl
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Repeat the previous step (step 7.5).
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about  , but do not dry the pellet to the point of cracking.
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Remove the tubes from the magnetic rack and resuspended each pellet in  nuclease-free water. Incubate for  at  .
15 µl
0 Room temperature
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Remove and retain  of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.
15 µl
?. [PCR amplicon cleanup and concentrate (400bp & 1200bp)]
Quantify   of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.
1 µl
This is a safe stop point.For short-term storage, samples can be stored at    overnight;For long-term storage, samples should be stored at   .
?. [DNA End-Prep (nanopore)]
Thaw the NEBNext Ultra II End repair / dA-tailing Module reagents on ice.
?. [Native barcode ligation and cleanup (nanopore)]
Thaw the native barcodes at room temperature, enough for one barcode per sample. Individually mix the barcodes by pipetting, and place them  .Thaw the tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place  .
on ice
on ice
?. [Adapter ligation and cleanup (nanopore)]
Thaw the Elution Buffer (EB), Short Fragment Buffer (SFB), and NEBNext Quick Ligation Reaction Buffer (5x) at  , mix by vortexing, spin down and place  . Check the contents of each tube are clear of any precipitate.Spin down the T4 Ligase and the Adapter Mix II (AMII), and place .
0 Room temperature
on ice
on ice
?. [Priming and loading the SpotON flowcell (nanopore)]
Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at .Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by vortexing and spin down at  .
0 Room temperature
0 Room temperature
?. [DNA End-Prep (nanopore)]
Determine the volume of the cleaned-up PCR reaction that yields  () of DNA per sample and aliquot in new 0.5 mL PCR tubes.
200
50 ng
?. [DNA End-Prep (nanopore)]
Make up each sample per tube to   using nuclease-free water.
12.5 µl
?. [DNA End-Prep (nanopore)]
Prepare end-prep mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place  .ComponentVolumeUltra II End-prep reaction buffer1.75 μlUltra II End-prep enzyme mix0.75 μlTotal2.5 μl
on ice
ComponentVolumeUltra II End-prep reaction buffer1.75 μlUltra II End-prep enzyme mix0.75 μlTotal2.5 μl
?. [DNA End-Prep (nanopore)]
Add  end-prep mastermix to each tube, mix by pipetting or flicking the tube.
2.5 µl
?. [DNA End-Prep (nanopore)]
Using a thermal cycler, incubate at   for  and  for
20 °C
65 °C
?. [Native barcode ligation and cleanup (nanopore)]
Add the reagents in the following order per tube, mixing by flicking the tube between each sequential addition (one barcode per sample):ComponentVolumeNuclease-free water6 μlEnd-prepped DNA1.5 μlNative Barcode2.5 μlBlunt/TA Ligase Master Mix10 μlTotal20 μl
ComponentVolumeNuclease-free water6 μlEnd-prepped DNA1.5 μlNative Barcode2.5 μlBlunt/TA Ligase Master Mix10 μlTotal20 μl
?. [Native barcode ligation and cleanup (nanopore)]
Mix contents thoroughly by pipetting or flcking the tube and spin down briefly.
?. [Native barcode ligation and cleanup (nanopore)]
Using a thermal cycler, incubate at   for  and at   for  .
20 °C
65 °C
?. [Native barcode ligation and cleanup (nanopore)]
Pool each barcoded library into a new 1.5 Eppendorf DNA LoBind tube.
?. [Native barcode ligation and cleanup (nanopore)]
Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.
?. [Native barcode ligation and cleanup (nanopore)]
Add 0.4x volume of pooled barcoded reaction of resuspended Mag-Bind® TotalPure NGS beads to the pooled barcoded reaction. Briefly vortex to mix and spin down.
?. [Native barcode ligation and cleanup (nanopore)]
Incubate on a Hula mixer (rotator mixer) at  for  .
0 Room temperature
?. [Native barcode ligation and cleanup (nanopore)]
Prepare  fresh 80% ethanol in nuclease-free water.
500 µl
?. [Native barcode ligation and cleanup (nanopore)]
Spin down and pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
?. [Native barcode ligation and cleanup (nanopore)]
Wash the beads by adding  Short Fragment Buffer (SFB). Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
700 µl
?. [Native barcode ligation and cleanup (nanopore)]
Repeat the previous step (step 11.6).
?. [Native barcode ligation and cleanup (nanopore)]
Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about  , but do not dry the pellet to the point of cracking.
?. [Native barcode ligation and cleanup (nanopore)]
Remove the tubes from the magnetic rack and resuspended each pellet in  nuclease-free water. Incubate for  at  .
35 µl
0 Room temperature
?. [Native barcode ligation and cleanup (nanopore)]
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
?. [Native barcode ligation and cleanup (nanopore)]
Remove and retain  of eluate into a new 1.5 mL Eppendorf tubes.
35 µl
?. [Native barcode ligation and cleanup (nanopore)]
Keep the tubes on the magnet and wash the beads with   of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
500 µl
?. [Native barcode ligation and cleanup (nanopore)]
Quantify   of eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.
1 µl
?. [Adapter ligation and cleanup (nanopore)]
Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.ComponentVolumePooled barcoded sample30 μlAdapter Mix II (AMII)5 μlNEBNext Quick Ligation Reaction Buffer (5X)10 μlQuick T4 DNA Ligase5 μlTotal50 μl
ComponentVolumePooled barcoded sample30 μlAdapter Mix II (AMII)5 μlNEBNext Quick Ligation Reaction Buffer (5X)10 μlQuick T4 DNA Ligase5 μlTotal50 μl
?. [Adapter ligation and cleanup (nanopore)]
Mix gently by flicking the tube, and spin down.
?. [Adapter ligation and cleanup (nanopore)]
Incubate the reaction for  at room temperature.
?. [Adapter ligation and cleanup (nanopore)]
Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.
?. [Adapter ligation and cleanup (nanopore)]
Add  (0.4x volume) of resuspended Mag-Bind® TotalPure NGS beads to the reaction. Briefly vortex to mix and spin down.
20 µl
?. [Adapter ligation and cleanup (nanopore)]
Incubate on a Hula mixer (rotator mixer) at  for  .
0 Room temperature
?. [Adapter ligation and cleanup (nanopore)]
Spin down and pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
?. [Adapter ligation and cleanup (nanopore)]
Wash the beads by adding  Short Fragment Buffer (SFB). Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
125 µl
?. [Adapter ligation and cleanup (nanopore)]
Repeat the previous step (step 14.4).
?. [Adapter ligation and cleanup (nanopore)]
Spin down and place the tubes on the magnet. Pipette off any residual supernatant.
?. [Adapter ligation and cleanup (nanopore)]
Remove the tubes from the magnetic rack and resuspended each pellet in  Elution Buffer (EB). Incubate for  at  .
15 µl
0 Room temperature
?. [Adapter ligation and cleanup (nanopore)]
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
?. [Adapter ligation and cleanup (nanopore)]
Remove and retain  of eluate into a new 1.5 mL Eppendorf tubes.
15 µl
?. [Adapter ligation and cleanup (nanopore)]
Quantify   of eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.
1 µl
?. [Priming and loading the SpotON flowcell (nanopore)]
Open the MinION Mk1B lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.
?. [Priming and loading the SpotON flowcell (nanopore)]
Slide the priming port cover clockwise to open the priming port.
?. [Priming and loading the SpotON flowcell (nanopore)]
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl).
?. [Priming and loading the SpotON flowcell (nanopore)]
To prepare the flow cell priming mix, add  of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at  .
30 µl
0 Room temperature
?. [Priming and loading the SpotON flowcell (nanopore)]
Load  of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for  . During this time, prepare the library for loading by following the steps below.
800 µl
?. [Priming and loading the SpotON flowcell (nanopore)]
Thoroughly mix the contents of the Loading Beads (LB) by pipetting.
?. [Priming and loading the SpotON flowcell (nanopore)]
In a new tube, prepare the library for loading as follows:ReagentVolumeSequencing Buffer (SQB)37.5 μlLoading Beads (LB), mixed immediately before use25.5 μlDNA library (50 fmol)12 μlTotal75 μl
ReagentVolumeSequencing Buffer (SQB)37.5 μlLoading Beads (LB), mixed immediately before use25.5 μlDNA library (50 fmol)12 μlTotal75 μl
?. [Priming and loading the SpotON flowcell (nanopore)]
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
?. [Priming and loading the SpotON flowcell (nanopore)]
Load  of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
200 µl
?. [Priming and loading the SpotON flowcell (nanopore)]
Mix the prepared library gently by pipetting up and down just prior to loading.
?. [Priming and loading the SpotON flowcell (nanopore)]
Add  of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
75 µl
?. [Priming and loading the SpotON flowcell (nanopore)]
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION Mk1B lid.
?. [PCR amplicon cleanup and concentrate (150bp)]
Combine the  PCR reaction mixtures for the two pools per sample into new 1.5 mL Eppendorf tubes, one per sample.
25 µl
?. [PCR amplicon cleanup and concentrate (150bp)]
Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.
?. [PCR amplicon cleanup and concentrate (150bp)]
Add  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at  for  .
[(0.85x beads/sample ratio)]
0 Room temperature
?. [PCR amplicon cleanup and concentrate (150bp)]
Pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, pipette off the supernatant (keep beads).
?. [PCR amplicon cleanup and concentrate (150bp)]
Prepare fresh 80% ethanol in nuclease-free water that enough for  per sample.
400 µl
?. [PCR amplicon cleanup and concentrate (150bp)]
Pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, pipette   supernatant to new 1.5 mL Eppendorf tubes (keep supernatant).
92.5 µl
?. [PCR amplicon cleanup and concentrate (150bp)]
Keep the tubes on the magnet and wash the beads with   of freshly prepared 80% ethanol without disturbing the pellet. Incubate for  and pipette off the ethanol.
200 µl
?. [PCR amplicon cleanup and concentrate (150bp)]
Repeat the previous step (step 17.7).
?. [PCR amplicon cleanup and concentrate (150bp)]
Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about  , but do not dry the pellet to the point of cracking.
?. [PCR amplicon cleanup and concentrate (150bp)]
Remove the tubes from the magnetic rack and resuspended each pellet in  nuclease-free water. Incubate for  at  .
15 µl
0 Room temperature
?. [PCR amplicon cleanup and concentrate (150bp)]
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
?. [PCR amplicon cleanup and concentrate (150bp)]
Remove and retain  of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.
15 µl
?. [PCR amplicon cleanup and concentrate (150bp)]
Add  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at  for  .
[(1.8x total beads/sample ratio)]
0 Room temperature
?. [DNA End-Prep (Illumina)]
Thaw the NEBNext Ultra II End repair / dA-tailing Module reagents on ice.
?. [DNA End-Prep (Illumina)]
Determine the volume of the cleaned-up PCR reaction that yields  of DNA per sample and aliquot in new 0.5 mL PCR tubes.
100 ng
?. [DNA End-Prep (Illumina)]
Make up each sample per tube to   using nuclease-free water.
20 µl
?. [DNA End-Prep (Illumina)]
Prepare end-prep mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place  .ComponentVolumeUltra II End-prep reaction buffer2.8 μlUltra II End-prep enzyme mix1.2 μlTotal4 μl
on ice
ComponentVolumeUltra II End-prep reaction buffer2.8 μlUltra II End-prep enzyme mix1.2 μlTotal4 μl
?. [DNA End-Prep (Illumina)]
Add  end-prep mastermix to each tube, mix by pipetting or flicking the tube.
4 µl
?. [DNA End-Prep (Illumina)]
Using a thermal cycler, incubate at   for  and  for  , then hold at  .
20 °C
65 °C
4 °C
?. [Adapter ligation and cleanup (Illumina)]
Dilute the NEBNext Adaptor for Illumina 10x in Tris/NaCl, pH 7.5-8.0.
?. [Adapter ligation and cleanup (Illumina)]
Prepare adapter ligation mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place ComponentVolume (µl)NEBNext Adaptor for Illumina1NEBNext Ultra II Ligation Master Mix12NEBNext Ligation Enhancer0.4Total Volume13.4
on ice
ComponentVolume (µl)NEBNext Adaptor for Illumina1NEBNext Ultra II Ligation Master Mix12NEBNext Ligation Enhancer0.4Total Volume13.4
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C.
?. [Adapter ligation and cleanup (Illumina)]
Add  1adapter ligation mastermix directly to the  End Prep Reaction Mixture, mix by pipetting or flicking the tubes and spin down.
13.4 µl
24 µl
?. [Adapter ligation and cleanup (Illumina)]
Incubate at  for  in a thermocycler with the heated lid off (or open lid).
20 °C
?. [Adapter ligation and cleanup (Illumina)]
Add  11.2µl of USER® Enzyme to the ligation mixture.
1.2 µl
?. [Adapter ligation and cleanup (Illumina)]
Mix well and incubate in a thermocycler at  for .
37 °C
?. [Adapter ligation and cleanup (Illumina)]
Transfer the  ligation reaction mix into new 1.5 mL Eppendorf tubes, one per sample.
36 µl
?. [Adapter ligation and cleanup (Illumina)]
Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.
?. [Adapter ligation and cleanup (Illumina)]
Add  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at  for  .
[(1.2x beads/sample ratio)]
0 Room temperature
?. [Adapter ligation and cleanup (Illumina)]
Prepare fresh 80% ethanol in nuclease-free water that enough for  per sample.
400 µl
?. [Adapter ligation and cleanup (Illumina)]
Pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
?. [Adapter ligation and cleanup (Illumina)]
Keep the tubes on the magnet and wash the beads with   of freshly prepared 80% ethanol without disturbing the pellet. Incubate for  and pipette off the ethanol.
200 µl
?. [Adapter ligation and cleanup (Illumina)]
Repeat the previous step (step 21.5).
?. [Adapter ligation and cleanup (Illumina)]
Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about  , but do not dry the pellet to the point of cracking.
?. [Adapter ligation and cleanup (Illumina)]
Remove the tubes from the magnetic rack and resuspended each pellet in  nuclease-free water. Incubate for  at  .
15 µl
0 Room temperature
?. [Adapter ligation and cleanup (Illumina)]
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
?. [Adapter ligation and cleanup (Illumina)]
Remove and retain  of eluate containing the DNA library per tube into new 0.5 mL PCR tubes.
15 µl
?. [PCR amplicon cleanup and concentrate (150bp)]
Quantify   of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.
1 µl
This is a safe stop point.For short-term storage, samples can be stored at    overnight;For long-term storage, samples should be stored at   .
?. [PCR Barcoding and cleanup(Illumina)]
Prepare barcoding mastermix, mix by pipetting or flicking the tube, spin down and place ComponentVolume (µl)NEBNext Ultra II Q5 Master Mix25Index Primer (one per sample)5Universal PCR Primer5Total Volume35
on ice
ComponentVolume (µl)NEBNext Ultra II Q5 Master Mix25Index Primer (one per sample)5Universal PCR Primer5Total Volume35
?. [PCR Barcoding and cleanup(Illumina)]
Add  barcoding mastermix to  cleaned adapter ligation mixture, mix well by pipetting or flicking the tubes.
35 µl
15 µl
?. [PCR Barcoding and cleanup(Illumina)]
Run in a thermal cycler using the following program:StepTemperatureTimeCyclesInitial denaturation  98 °C30 s1Denaturation Annealing/Extension98 °C                 65 °C10 s                   75 s4Final extension65 °C5 min1Hold4 °C ∞1
StepTemperatureTimeCyclesInitial denaturation  98 °C30 s1Denaturation Annealing/Extension98 °C                 65 °C10 s                   75 s4Final extension65 °C5 min1Hold4 °C ∞1
?. [PCR Barcoding and cleanup(Illumina)]
Pool equal mass of each barcoded library to form a pooled barcoded library.
?. [PCR Barcoding and cleanup(Illumina)]
Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.
?. [PCR Barcoding and cleanup(Illumina)]
Add 1.0x volume of pooled barcoded reaction of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down.
?. [PCR Barcoding and cleanup(Illumina)]
Incubate at  for  .
0 Room temperature
?. [PCR Barcoding and cleanup(Illumina)]
Prepare fresh 80% ethanol in nuclease-free water that enough for  per sample.
400 µl
?. [PCR Barcoding and cleanup(Illumina)]
Pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
?. [PCR Barcoding and cleanup(Illumina)]
Keep the tubes on the magnet and wash the beads with   of freshly prepared 80% ethanol without disturbing the pellet. Incubate for  and pipette off the ethanol.
200 µl
?. [PCR Barcoding and cleanup(Illumina)]
Repeat the previous step (step 25.6).
?. [PCR Barcoding and cleanup(Illumina)]
Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about  , but do not dry the pellet to the point of cracking.
?. [PCR Barcoding and cleanup(Illumina)]
Remove the tubes from the magnetic rack and resuspended each pellet in  nuclease-free water. Incubate for  at  .
20 µl
0 Room temperature
?. [PCR Barcoding and cleanup(Illumina)]
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
?. [PCR Barcoding and cleanup(Illumina)]
Remove and retain  of eluate containing the DNA library per tube into new 0.5 mL PCR tubes.
20 µl
?. [PCR Barcoding and cleanup(Illumina)]
Transfer   barcoded library into new 1.5 mL Eppendorf tubes, one per sample. Add  nuclease-free water to each tube.
20 µl
30 µl
?. [PCR Barcoding and cleanup(Illumina)]
Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.
?. [PCR Barcoding and cleanup(Illumina)]
Add  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at  for  .
[(0.7x beads/sample ratio)]
0 Room temperature
?. [PCR Barcoding and cleanup(Illumina)]
Prepare fresh 80% ethanol in nuclease-free water that enough for  per sample.
400 µl
?. [PCR Barcoding and cleanup(Illumina)]
Pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, pipette   supernatant to new 1.5 mL Eppendorf tubes (keep supernatant).
85 µl
?. [PCR Barcoding and cleanup(Illumina)]
Add  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at  for  .
[(1.0x total beads/sample ratio)]
0 Room temperature
?. [PCR Barcoding and cleanup(Illumina)]
Pellet the beads on a magnet rack for  . Keep the tubes on the magnet until the eluate is clear and colourless, pipette off the supernatant (keep beads).
?. [PCR Barcoding and cleanup(Illumina)]
Keep the tubes on the magnet and wash the beads with   of freshly prepared 80% ethanol without disturbing the pellet. Incubate for  and pipette off the ethanol.
200 µl
?. [PCR Barcoding and cleanup(Illumina)]
Repeat the previous step (step 23.7).
?. [PCR Barcoding and cleanup(Illumina)]
Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about  , but do not dry the pellet to the point of cracking.
?. [PCR Barcoding and cleanup(Illumina)]
Remove the tubes from the magnetic rack and resuspended each pellet in  nuclease-free water. Incubate for  at  .
20 µl
0 Room temperature
?. [PCR Barcoding and cleanup(Illumina)]
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
?. [PCR Barcoding and cleanup(Illumina)]
Remove and retain  of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.
20 µl
?. [PCR Barcoding and cleanup(Illumina)]
Quantify   of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.
1 µl
This is a safe stop point.For short-term storage, samples can be stored at    overnight;For long-term storage, samples should be stored at   .
?. [PCR Barcoding and cleanup(Illumina)]
Quantify   of pooled barcoded library using a Qubit fluorometer with Qubit dsDNA HS Assay Kit. This sequencing library is ready for submission.
1 µl
This is a safe stop point.Store this sequencing library at   .