Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater

Xuan  Lin; Melissa   Glier; Kevin Kuchinski; Tenysha Ross-Van Mierlo; David McVea; John  Tyson; Natalie Prystajecky; Ryan  Ziels

Sep 30, 2021

Version 1

Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater V.1

DOI

dx.doi.org/10.17504/protocols.io.buccnssw

Xuan Lin¹,
Melissa Glier²,
Kevin Kuchinski^2,3,
Tenysha Ross-Van Mierlo⁴,
David McVea⁵,
John Tyson²,
Natalie Prystajecky^2,3,
Ryan Ziels¹

¹Civil Engineering, University of British Columbia, Vancouver, BC, Canada;
²Environmental Microbiology, British Columbia Center for Disease Control Public Health Laboratory, Vancouver, BC, Canada;
³Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada;
⁴Biological Sciences, Simon Fraser University, Burnaby, BC, Canada;
⁵Environmental Health Services, British Columbia Center for Disease Control Public Health Laboratory, Vancouver, BC, Canada

Xuan Lin

University of British Columbia

DOI: dx.doi.org/10.17504/protocols.io.buccnssw

Protocol Citation: Xuan Lin, Melissa Glier, Kevin Kuchinski, Tenysha Ross-Van Mierlo, David McVea, John Tyson, Natalie Prystajecky, Ryan Ziels 2021. Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater. protocols.io https://dx.doi.org/10.17504/protocols.io.buccnssw

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it’s working

Created: April 19, 2021

Last Modified: September 30, 2021

Protocol Integer ID: 49252

Abstract

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.

This protocol is adapted from the Swift amplicon™ SARS-COV-2 protocol (150bp amplicon),  ARTIC V3 protocol (400bp amplicon), and "midnight" protocol (1200bp amplicon).

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®.

Links to the protocols are:
Swift amplicon protocol (150bp): https://swiftbiosci.com/swift-amplicon-sars-cov-2-panel/
ARTIC V3 protocol (400bp): https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye?version_warning=no
"midnight" protocol V4 (1200bp): dx.doi.org/10.17504/protocols.io.bh7hj9j6

Materials

Primers
Swift primers were purchased as two pools form IDT
ReagentSARS-CoV2-Midnight-1200 500rxnIDTCatalog #10007184  
ReagentARTIC nCoV-2019 V3 PanelIDT  

Reverse Transcription
ReagentSuperScript™ IV First-Strand Synthesis SystemInvitrogen - Thermo FisherCatalog #18091200  
cDNA cleanup
ReagentDNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003  
Multiplex PCR
ReagentQ5® Hot Start High-Fidelity 2X Master MixNew England BiolabsCatalog #M0494L  
ReagentMag-Bind® TotalPure NGS beadsOmega BiotekCatalog #M1378-01  
Oxford Nanopore Sequencing

ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S  
ReagentBlunt/TA Ligase Master Mix - 250 rxnsNew England BiolabsCatalog #M0367L  ReagentNative Barcoding Expansion 1-12 (PCR-free)Catalog #EXP-NBD104  ReagentNative Barcoding Expansion 13-24 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD114  ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S    ReagentSFB expansionOxford Nanopore TechnologiesCatalog #EXP-SFB001  ReagentLigation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK109  ReagentONT MinION Flow Cell R9.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN106D  
Illumina Sequencing
ReagentNEBNext Ultra II DNA Library Prep Kit for Illumina - 24 rxnsNew England BiolabsCatalog #E7645S  ReagentNEBNext Multiplex Oligos for Illumina (Index Primers Set 1) - 24 rxnsNew England BiolabsCatalog #E7335S  

Protocol materials

ReagentSuperScript™ IV First-Strand Synthesis SystemInvitrogen - Thermo FisherCatalog #18091200 
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S 
ReagentSARS-CoV2-Midnight-1200 500rxnIDTCatalog #10007184 
ReagentARTIC nCoV-2019 V3 PanelIDT 
ReagentMag-Bind® TotalPure NGS beadsOmega BiotekCatalog #M1378-01 
ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S 
ReagentSFB expansionOxford Nanopore TechnologiesCatalog #EXP-SFB001 
ReagentLigation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK109 
ReagentNEBNext Ultra II DNA Library Prep Kit for Illumina - 24 rxnsNew England BiolabsCatalog #E7645S 
ReagentQ5® Hot Start High-Fidelity 2X Master MixNew England BiolabsCatalog #M0494L 
ReagentBlunt/TA Ligase Master Mix - 250 rxnsNew England BiolabsCatalog #M0367L 
ReagentONT MinION Flow Cell R9.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN106D 
ReagentNEBNext Multiplex Oligos for Illumina (Index Primers Set 1) - 24 rxnsNew England BiolabsCatalog #E7335S 
ReagentDNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003 
ReagentNative Barcoding Expansion 1-12 (PCR-free)Catalog #EXP-NBD104 
ReagentNative Barcoding Expansion 13-24 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD114 
ReagentDNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003 
ReagentZymo DNA Binding BufferZymo ResearchCatalog #D4003-1-25 
ReagentZymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6 
ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1 

Before start

Time can be saved by preparing master mixes first, before PCR steps. The master mix for cDNA and PCR steps should be prepared in Master Mix (PCR) Hood. To avoid cross-contamination make sure that your original stock reagents have no contact with RNA or any amplified DNA material.

A Negative Control (nuclease-free H2O ) should be included from cDNA synthesis step until the end.
Keep the enzymes on ice and thaw the other reagents at room temperature before placing on ice. 

Sequencing library preparation should be performed in post-PCR area.

First strand cDNA synthesis

1h 30m

Preparation of master mix

Prepare the following components in a 1.5 mL Eppendorf DNA LoBind tube for the number of samples that will be tested and dispense Amount4 µL Master Mix RT_1  to 0.2mL each PCR tubes. Briefly spin down the 0.2 mL tubes containing Master Mix RT_1 and keep TemperatureOn ice   

AB
ComponentVolume
50µM random hexamers2 μL
10mM dNTPs mix (10mM each)2 μL

Prepare the following components in a 1.5mL tube and keep the Master Mix RT_2 TemperatureOn ice   . 


AB
ComponentVolume (μL)
100 mM DTT2
Ribonuclease Inhibitor (40 U/µL)2
5x SSIV Buffer8
SSIV Reverse transcriptase2
Nuclease free H2O10

This step should be conducted in the pre-PCR area (e.g. cleaned DNA hood). Keep all the Master Mix TemperatureOn ice  while doing this step. 

Add Amount12 µL RNA extract  to each 0.2 mL tube containing the Master Mix RT_1

Mix by pipetting or flicking the tube, spin down briefly.

Incubate the reaction mix in a thermocycler at Temperature65 °C   for Duration00:05:00   , then spin down briefly, and incubate immediately TemperatureOn ice   for at least Duration00:01:00   .

While on ice, add to each tube Amount24 µL Master Mix RT_2 ,

Gently mix by pipetting or flicking the tubes and briefly spin down.

Incubate the reaction mix in the thermocycler for:


ABC
  Step    Temp    Time  
  1    42 °C    00:50:00   
  2    70 °C    00:10:00      
  3    4°C    hold  


Note
The cDNA can be stored at Temperature-20 °C   . If needed, it is a safe stop point. The remaining RNA should be stored at Temperature-80 °C   .

cDNA cleanup and concentration

cDNA cleanup withReagentDNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003  (all centrifuge steps at 13,000xg)

Add Amount8 µL 0.5M EDTA   and Amount8 µL 1N NaOH  to the Amount40 µL RT reaction  .

Incubate the reaction mix in a thermocycler at Temperature65 °C   for Duration00:15:00    to hydrolyze RNA.

15m

Transfer the hydrolysis reaction mix from the last step to a new 1.5 mL Eppendorf DNA LoBind tube and add Amount392 µL (7x volume)   ReagentZymo DNA Binding BufferZymo ResearchCatalog #D4003-1-25  to Amount56 µL hydrolysis reaction mix .  Briefly vortex to mix, and pulse spin to collect thesample.

Transfer mixture to a provided Zymo-Spin™ Column in a Collection Tube.

Centrifuge for Duration00:00:30   and discard the flow-through.

30s

Add Amount200 µL   ReagentZymo DNA Wash BufferZymo ResearchCatalog #D4003-2-6 to the column. Centrifuge for Duration00:00:30   .

30s

Repeat step 3.6.

Add ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1 to the column matrix and incubate at TemperatureRoom temperature    for Duration00:05:00   .


AB
Primer SchemeElute volume (μL)
Swift_150bp14
ARTIC V3_400bp18.5
Midnight_1200bp25

Transfer the column to a 1.5 ml microcentrifuge tube, then centrifuge for Duration00:00:30    to elute the DNA and keep on ice.

30s

Multiplex PCR

Prepare the following PCR mastermixs and keep TemperatureOn ice   . 

Prepare PCR mastermix for Swift_150bp primer scheme.

ABC
ComponentReaction 1Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µL
Swift Pool 1 (15nM each primer)6.25 μL0
Swift Pool 2 (15nM each primer)06.25 μL
Total18.75 μL18.75 μL

Prepare PCR mastermix for ARTIC V3_400bp primer scheme.

ABC
ComponentReaction 1Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µL
V3 Pool 1 (15nM each primer)4 μL0
V3 Pool 2 (15nM each primer)04 μL
Total16.5 μL16.5 μL

Prepare PCR mastermix for midnight_1200bp primer scheme.

ABC
ComponentReaction 1Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µL
midnight Pool 1 (15nM each primer)1.1 μL0
midnight Pool 2 (15nM each primer)01.1 μL
Total13.6 μL13.6 μL

Add the corresponding amount of cDNA to each of the PCR reactions, mix by pipetting or flicking the tube and spin down.

AB
Primer SchemeVolume per reaction (μL)
Swift_150bp6.25
ARTIC V3_400bp8.5
Midnight_1200bp11.4

Run in a thermal cycler using the following program:

Swift_150bp primer scheme
ABCD
StepTemperatureTimeCycles
Initial denaturation  98 °C30 s1
Denaturation  Annealing/Extension  98°C                   65°C15 s                    2 min35
Hold4 °C ∞1

OR

ARTIC V3 and midnight primer scheme
ABCD
StepTemperatureTimeCycles
Initial denaturation  98 °C30 s1
Denaturation  Annealing/Extension  98°C                   65°C15 s                    5 min35
Hold4 °C ∞1

Note
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)

30m

Combine the Amount25 µL   PCR reaction mixtrues for the two pools per sample into new 1.5 mL Eppendorf tubes, one per sample.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Add Amount50 µL   of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down.
Incubate at TemperatureRoom temperature   for Duration00:05:00   .

Prepare fresh 80% ethanol in nuclease-free water that enough for Amount400 µL   per sample.

Pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with Amount200 µL    of freshly prepared 80% ethanol without disturbing the pellet. Incubate for Duration00:00:30   and pipette off the ethanol.

30s

Repeat the previous step (step 7.5).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about Duration00:00:30   , but do not dry the pellet to the point of cracking.

30s

Remove the tubes from the magnetic rack and resuspended each pellet in Amount15 µL   nuclease-free water. Incubate for Duration00:05:00   at TemperatureRoom temperature   .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain Amount15 µL   of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.

Quantify Amount1 µL    of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Note
This is a safe stop point.
For short-term storage, samples can be stored at Temperature4 °C    overnight;
For long-term storage, samples should be stored at Temperature-20 °C   .

DNA End-Prep (nanopore)

20m

Thaw the NEBNext Ultra II End repair / dA-tailing Module reagents on ice.

Determine the volume of the cleaned-up PCR reaction that yields Amount200 fmol   (Amount50 ng  ) of DNA per sample and aliquot in new 0.5 mL PCR tubes.

Make up each sample per tube to Amount12.5 µL    using nuclease-free water.

Prepare end-prep mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place TemperatureOn ice   .

AB
ComponentVolume
Ultra II End-prep reaction buffer1.75 μl
Ultra II End-prep enzyme mix0.75 μl
Total2.5 μl

Add Amount2.5 µL   end-prep mastermix to each tube, mix by pipetting or flicking the tube.

Using a thermal cycler, incubate at Temperature20 °C    for Duration00:05:00   and Temperature65 °C   for Duration00:05:00   

10m

Native barcode ligation and cleanup (nanopore)

1h 30m

Thaw the native barcodes at room temperature, enough for one barcode per sample. Individually mix the barcodes by pipetting, and place them TemperatureOn ice   .

Thaw the tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place TemperatureOn ice   .

Add the reagents in the following order per tube, mixing by flicking the tube between each sequential addition (one barcode per sample):


AB
ComponentVolume
Nuclease-free water6 μl
End-prepped DNA1.5 μl
Native Barcode2.5 μl
Blunt/TA Ligase Master Mix10 μl
Total20 μl

Mix contents thoroughly by pipetting or flcking the tube and spin down briefly.

Using a thermal cycler, incubate at Temperature20 °C    for Duration00:20:00   and at Temperature65 °C    for Duration00:10:00   .

30m

Pool each barcoded library into a new 1.5 Eppendorf DNA LoBind tube.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

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.

Incubate on a Hula mixer (rotator mixer) at TemperatureRoom temperature   for Duration00:10:00   .

10m

Prepare Amount500 µL   fresh 80% ethanol in nuclease-free water.

Spin down and pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Wash the beads by adding Amount700 µL   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.

Repeat the previous step (step 11.6).

Keep the tubes on the magnet and wash the beads with Amount500 µL    of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about Duration00:00:30   , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in Amount35 µL   nuclease-free water. Incubate for Duration00:02:00   at TemperatureRoom temperature   .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain Amount35 µL   of eluate into a new 1.5 mL Eppendorf tubes.

Quantify Amount1 µL    of eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Adapter ligation and cleanup (nanopore)

1h 30m

Thaw the Elution Buffer (EB), Short Fragment Buffer (SFB), and NEBNext Quick Ligation Reaction Buffer (5x) at TemperatureRoom temperature   , mix by vortexing, spin down and place TemperatureOn ice   . Check the contents of each tube are clear of any precipitate.

Spin down the T4 Ligase and the Adapter Mix II (AMII), and place TemperatureOn ice  .

Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.


AB
ComponentVolume
Pooled barcoded sample30 μl
Adapter Mix II (AMII)5 μl
NEBNext Quick Ligation Reaction Buffer (5X)10 μl
Quick T4 DNA Ligase5 μl
Total50 μl

Mix gently by flicking the tube, and spin down.

Incubate the reaction for Duration00:20:00   at room temperature.

20m

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Add Amount20 µL   (0.4x volume) of resuspended Mag-Bind® TotalPure NGS beads to the reaction. Briefly vortex to mix and spin down.

Incubate on a Hula mixer (rotator mixer) at TemperatureRoom temperature   for Duration00:10:00   .

Spin down and pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Wash the beads by adding Amount125 µL   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.

Repeat the previous step (step 14.4).

Spin down and place the tubes on the magnet. Pipette off any residual supernatant.

Remove the tubes from the magnetic rack and resuspended each pellet in Amount15 µL   Elution Buffer (EB). Incubate for Duration00:05:00   at TemperatureRoom temperature   .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain Amount15 µL   of eluate into a new 1.5 mL Eppendorf tubes.

Quantify Amount1 µL    of eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Priming and loading the SpotON flowcell (nanopore)

30m

Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at TemperatureRoom temperature  .

Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by vortexing and spin down at TemperatureRoom temperature   .

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.

Slide the priming port cover clockwise to open the priming port.

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).

To prepare the flow cell priming mix, add Amount30 µL   of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at TemperatureRoom temperature   .

Load Amount800 µL   of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for Duration00:05:00   . During this time, prepare the library for loading by following the steps below.

Thoroughly mix the contents of the Loading Beads (LB) by pipetting.

In a new tube, prepare the library for loading as follows:

AB
ReagentVolume
Sequencing Buffer (SQB)37.5 μl
Loading Beads (LB), mixed immediately before use25.5 μl
DNA library (50 fmol)12 μl
Total75 μl

Gently lift the SpotON sample port cover to make the SpotON sample port accessible.

Load Amount200 µL   of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

Mix the prepared library gently by pipetting up and down just prior to loading.

Add Amount75 µL   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.

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)

45m

Combine the Amount25 µL   PCR reaction mixtures for the two pools per sample into new 1.5 mL Eppendorf tubes, one per sample.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Add Amount42.5 µL (0.85x beads/sample ratio)  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at TemperatureRoom temperature   for Duration00:05:00   .

Prepare fresh 80% ethanol in nuclease-free water that enough for Amount400 µL   per sample.

Pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, pipette Amount92.5 µL    supernatant to new 1.5 mL Eppendorf tubes (keep supernatant).

Add Amount47.5 µL (1.8x total beads/sample ratio)  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at TemperatureRoom temperature   for Duration00:15:00   .

15m

Pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, pipette off the supernatant (keep beads).

Keep the tubes on the magnet and wash the beads with Amount200 µL    of freshly prepared 80% ethanol without disturbing the pellet. Incubate for Duration00:00:30   and pipette off the ethanol.

Repeat the previous step (step 17.7).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about Duration00:00:30   , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in Amount15 µL   nuclease-free water. Incubate for Duration00:05:00   at TemperatureRoom temperature   .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain Amount15 µL   of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.

Quantify Amount1 µL    of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Note
This is a safe stop point.
For short-term storage, samples can be stored at Temperature4 °C    overnight;
For long-term storage, samples should be stored at Temperature-20 °C   .

DNA End-Prep (Illumina)

Thaw the NEBNext Ultra II End repair / dA-tailing Module reagents on ice.

Determine the volume of the cleaned-up PCR reaction that yields Amount100 ng   of DNA per sample and aliquot in new 0.5 mL PCR tubes.

Make up each sample per tube to Amount20 µL    using nuclease-free water.

Prepare end-prep mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place TemperatureOn ice   .

AB
ComponentVolume
Ultra II End-prep reaction buffer2.8 μl
Ultra II End-prep enzyme mix1.2 μl
Total4 μl

Add Amount4 µL   end-prep mastermix to each tube, mix by pipetting or flicking the tube.

Using a thermal cycler, incubate at Temperature20 °C    for Duration00:30:00   and Temperature65 °C   for Duration00:30:00   , then hold at Temperature4 °C   .

Adapter ligation and cleanup (Illumina)

Dilute the NEBNext Adaptor for Illumina 10x in Tris/NaCl, pH 7.5-8.0.

Prepare adapter ligation mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place TemperatureOn ice   


AB
ComponentVolume (µl)
NEBNext Adaptor for Illumina1
NEBNext Ultra II Ligation Master Mix12
NEBNext Ligation Enhancer0.4
Total Volume13.4



Note
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C.  
 

Add Amount13.4 µL   1adapter ligation mastermix directly to the Amount24 µL   End Prep Reaction Mixture, mix by pipetting or flicking the tubes and spin down.

Incubate at Temperature20 °C   for Duration00:15:00   in a thermocycler with the heated lid off (or open lid). 

15m

Add Amount1.2 µL   11.2µl of USER® Enzyme to the ligation mixture.

Mix well and incubate in a thermocycler at Temperature37 °C   for Duration00:15:00  .

15m

Transfer the Amount36 µL   ligation reaction mix into new 1.5 mL Eppendorf tubes, one per sample.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Add Amount43.2 µL (1.2x beads/sample ratio)  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at TemperatureRoom temperature   for Duration00:05:00   .

Prepare fresh 80% ethanol in nuclease-free water that enough for Amount400 µL   per sample.

Pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with Amount200 µL    of freshly prepared 80% ethanol without disturbing the pellet. Incubate for Duration00:00:30   and pipette off the ethanol.

Repeat the previous step (step 21.5).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about Duration00:00:30   , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in Amount15 µL   nuclease-free water. Incubate for Duration00:05:00   at TemperatureRoom temperature   .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain Amount15 µL   of eluate containing the DNA library per tube into new 0.5 mL PCR tubes.

PCR Barcoding and cleanup(Illumina)

2h 30m

Prepare barcoding mastermix, mix by pipetting or flicking the tube, spin down and place TemperatureOn ice   


AB
ComponentVolume (µl)
NEBNext Ultra II Q5 Master Mix25
Index Primer (one per sample)5
Universal PCR Primer5
Total Volume35

Add Amount35 µL   barcoding mastermix to Amount15 µL   cleaned adapter ligation mixture, mix well by pipetting or flicking the tubes.

Run in a thermal cycler using the following program:

ABCD
StepTemperatureTimeCycles
Initial denaturation  98 °C30 s1
Denaturation Annealing/Extension98 °C                 65 °C10 s                   75 s4
Final extension65 °C5 min1
Hold4 °C ∞1

Transfer  Amount20 µL   barcoded library into new 1.5 mL Eppendorf tubes, one per sample. Add Amount30 µL   nuclease-free water to each tube.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Add Amount35 µL (0.7x beads/sample ratio)  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at TemperatureRoom temperature   for Duration00:05:00   .

Prepare fresh 80% ethanol in nuclease-free water that enough for Amount400 µL   per sample.

Pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, pipette Amount85 µL    supernatant to new 1.5 mL Eppendorf tubes (keep supernatant).

Add Amount15 µL (1.0x total beads/sample ratio)  of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at TemperatureRoom temperature   for Duration00:15:00   .

Pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, pipette off the supernatant (keep beads).

Keep the tubes on the magnet and wash the beads with Amount200 µL    of freshly prepared 80% ethanol without disturbing the pellet. Incubate for Duration00:00:30   and pipette off the ethanol.

Repeat the previous step (step 23.7).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about Duration00:00:30   , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in Amount20 µL   nuclease-free water. Incubate for Duration00:05:00   at TemperatureRoom temperature   .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain Amount20 µL   of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.

Quantify Amount1 µL    of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Note
This is a safe stop point.
For short-term storage, samples can be stored at Temperature4 °C    overnight;
For long-term storage, samples should be stored at Temperature-20 °C   .

Pool equal mass of each barcoded library to form a pooled barcoded library.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

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.

Incubate at TemperatureRoom temperature   for Duration00:05:00   .

Prepare fresh 80% ethanol in nuclease-free water that enough for Amount400 µL   per sample.

Pellet the beads on a magnet rack for Duration00:05:00   . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with Amount200 µL    of freshly prepared 80% ethanol without disturbing the pellet. Incubate for Duration00:00:30   and pipette off the ethanol.

Repeat the previous step (step 25.6).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about Duration00:00:30   , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in Amount20 µL   nuclease-free water. Incubate for Duration00:05:00   at TemperatureRoom temperature   .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain Amount20 µL   of eluate containing the DNA library per tube into new 0.5 mL PCR tubes.

Quantify Amount1 µL    of pooled barcoded library using a Qubit fluorometer with Qubit dsDNA HS Assay Kit. This sequencing library is ready for submission.

Note
This is a safe stop point.
Store this sequencing library at Temperature-20 °C   .

	A	B
	Component	Volume
	50µM random hexamers	2 μL
	10mM dNTPs mix (10mM each)	2 μL

	A	B
	Component	Volume (μL)
	100 mM DTT	2
	Ribonuclease Inhibitor (40 U/µL)	2
	5x SSIV Buffer	8
	SSIV Reverse transcriptase	2
	Nuclease free H2O	10

A	B	C
Step	Temp	Time
1	42 °C	00:50:00
2	70 °C	00:10:00
3	4°C	hold

	A	B
	Primer Scheme	Elute volume (μL)
	Swift_150bp	14
	ARTIC V3_400bp	18.5
	Midnight_1200bp	25

A	B	C
Component	Reaction 1	Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix	12.5 µL	12.5 µL
Swift Pool 1 (15nM each primer)	6.25 μL	0
Swift Pool 2 (15nM each primer)	0	6.25 μL
Total	18.75 μL	18.75 μL

	A	B
	Primer Scheme	Volume per reaction (μL)
	Swift_150bp	6.25
	ARTIC V3_400bp	8.5
	Midnight_1200bp	11.4

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denaturation Annealing/Extension	98°C 65°C	15 s 2 min	35
Hold	4 °C	∞	1

	A	B
	Component	Volume
	Ultra II End-prep reaction buffer	1.75 μl
	Ultra II End-prep enzyme mix	0.75 μl
	Total	2.5 μl

	A	B
	Component	Volume
	Nuclease-free water	6 μl
	End-prepped DNA	1.5 μl
	Native Barcode	2.5 μl
	Blunt/TA Ligase Master Mix	10 μl
	Total	20 μl

	A	B
	Component	Volume
	Pooled barcoded sample	30 μl
	Adapter Mix II (AMII)	5 μl
	NEBNext Quick Ligation Reaction Buffer (5X)	10 μl
	Quick T4 DNA Ligase	5 μl
	Total	50 μl

	A	B
	Reagent	Volume
	Sequencing Buffer (SQB)	37.5 μl
	Loading Beads (LB), mixed immediately before use	25.5 μl
	DNA library (50 fmol)	12 μl
	Total	75 μl

	A	B
	Component	Volume (µl)
	NEBNext Adaptor for Illumina	1
	NEBNext Ultra II Ligation Master Mix	12
	NEBNext Ligation Enhancer	0.4
	Total Volume	13.4

	A	B
	Component	Volume (µl)
	NEBNext Ultra II Q5 Master Mix	25
	Index Primer (one per sample)	5
	Universal PCR Primer	5
	Total Volume	35

Public workspaceAssessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater V.1

Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater V.1