Mar 15, 2023

Public workspacePathogen-Oriented Low-cost Assembly & Re-sequencing on Opentrons (POLARtron): An automation-friendly highly sensitive and high-throughput SARS-CoV-2 diagnostic based on whole genome sequencing V.2

DOI
dx.doi.org/10.17504/protocols.io.81wgbp8jqvpk/v2
  • Per A. Adastra1,2,3,
  • Neva C. Durand1,2,3,4,
  • Namita Mitra1,2,3,
  • Saul Godinez1,2,3,
  • Ragini Mahajan1,3,5,
  • Alyssa Blackburn1,2,3,
  • Zane Colaric1,2,3,
  • Joshua W. M. Theisen1,3,
  • David Weisz1,2,3,
  • Olga Dudchenko1,2,3,
  • Andreas Gnirke1,4,
  • Suhas S.P. Rao1,2,3,6,
  • Parwinder Kaur7,
  • Erez Lieberman Aiden1,2,3,8,
  • Aviva Presser Aiden1,2,9,10
  • 1The Center for Genome Architecture, Baylor College of Medicine, Houston, TX 77030, USA;
  • 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX;
  • 3Center for Theoretical Biological Physics, Rice University, Houston, TX 77030, USA;
  • 4Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA;
  • 5Department of Biosciences, Rice University, Houston, TX 77030, USA;
  • 6Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA;
  • 7UWA School of Agriculture and Environment, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia;
  • 8Departments of Computer Science and Computational and Applied Mathematics, Rice University, Houston, TX 77030, USA;
  • 9Department of Bioengineering, Rice University, Houston, TX, USA;
  • 10Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA
Per A. Adastra
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DOI: dx.doi.org/10.17504/protocols.io.81wgbp8jqvpk/v2
Protocol Citation: Per A. Adastra, Neva C. Durand, Namita Mitra, Saul Godinez, Ragini Mahajan, Alyssa Blackburn, Zane Colaric, Joshua W. M. Theisen, David Weisz, Olga Dudchenko, Andreas Gnirke, Suhas S.P. Rao, Parwinder Kaur, Erez Lieberman Aiden, Aviva Presser Aiden 2023. Pathogen-Oriented Low-cost Assembly & Re-sequencing on Opentrons (POLARtron): An automation-friendly highly sensitive and high-throughput SARS-CoV-2 diagnostic based on whole genome sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbp8jqvpk/v2Version created by Per A. Adastra
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: In development
We are still developing and optimizing this protocol
Created: March 15, 2023
Last Modified: March 15, 2023
Protocol Integer ID: 78863
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
An automation-friendly variant of POLAR.
Guidelines
SARS-CoV-2 Specific Primer Set

The ARTIC Network designed and tested1 the primer set used in this protocol and must be custom ordered prior to experiments. Details on their primer set can be found on their Github page https://github.com/artic-network/artic-ncov2019.

  1. Artic Network. https://artic.network/resources/ncov/ncov-amplicon-v3.pdf.

The World Health Organization: Dos and Don’ts for Molecular Testing
(https://www.who.int/malaria/areas/diagnosis/molecular-testing-dos-donts/en/)

Molecular detection methods have the ability to produce a large volume of nucleic acid through the amplification of trace quantities found in samples. While this is beneficial for enabling sensitive detection, it also introduces the possibility of contamination through the spreading of amplicon aerosols in the laboratory environment. When conducting experiments, measures can be undertaken to avoid the contamination of reagents, laboratory equipment, and bench space, as such contamination may generate false-positive (or false-negative) results.
To help reduce the likelihood of contamination, Good Laboratory Practice should be exercised at all times. Specifically, precautions should be taken regarding the following points:

Handling reagents

  • Briefly centrifuge reagent tubes before opening to avoid the generation of aerosols.
  • Aliquot reagents to avoid multiple freeze-thaw and the contamination of master stocks.
  • Clearly label and date all reagent and reaction tubes and maintain logs of reagent lot and batch numbers used in all experiments.
  • Pipette all reagents and samples using filter tips. Prior to purchase, it is advisable to confirm with the manufacturer that the filter tips fit the brand of the pipette to be used.


Organization of workspace and equipment


The workspace should be organized to ensure that the flow of work occurs in one direction, from clean areas (pre-PCR) to dirty areas (post-PCR). The following general precautions will help to reduce the chance of contamination.

Have separate designated rooms, or at minimum physically separate areas, for:
  1. master mix preparation,
  2. nucleic acid extraction and DNA template addition

In some settings, having 4 separate rooms is difficult. A possible but less desirable option is to do the master mix preparation in a containment area, e.g. a laminar flow cabinet. In the case of nested PCR amplification, the preparation of the master mix for the second round reaction should be prepared in the ‘clean’ area for master mix preparation, but the inoculation with the primary PCR product should be done in the amplification room, and if possible in a dedicated containment area (e.g. a laminar flow cabinet).

Each room/area needs a separate set of clearly labeled pipettes, filter tips, tube racks, vortexes, centrifuges (if relevant), pens, generic lab reagents, lab coats, and boxes of gloves that will remain at their respective workstations.
Hands must be washed and gloves and lab coats changed when moving between the designated areas. Reagents and equipment should not be moved from a dirty area to a clean area. Should an extreme case arise where a reagent or piece of equipment needs to be moved backward, it must first be decontaminated with 10% sodium hypochlorite, followed by a wipe down with sterile water

Ideally, staff should abide by the unidirectional workflow ethos and not go from dirty areas (post-PCR) back to clean areas (pre-PCR) on the same day. However, there may be occasions when this is unavoidable. When such occasion arises, personnel must take care to thoroughly wash hands, change gloves, use the designated lab coat and not introduce any equipment they will want to take out of the room again, such as lab books. Such control measures should be emphasized in staff training on molecular methods.

After use, bench spaces should be cleaned with 10% sodium hypochlorite (followed by sterile water to remove residual bleach), 70% ethanol, or a validated commercially available DNA-destroying decontaminant. Ideally, ultra-violet (UV) lamps should be fitted to enable decontamination by irradiation. However, the use of UV lamps should be restricted to closed working areas, e.g. safety cabinets, in order to limit the laboratory staff’s UV exposure. Please abide by manufacturer instructions for UV lamp care, ventilation, and cleaning in order to ensure that lamps remain effective.

If manufacturer instructions permit it, pipettes should be routinely sterilized by autoclave. If pipettes cannot be autoclaved, it should suffice to clean them with 10% sodium hypochlorite (followed by a thorough wipe down with sterile water) or with a commercial DNA-destroying decontaminant followed by UV exposure.

All equipment needs to be calibrated regularly according to the manufacturer-recommended schedule. A designated person should be in charge of ensuring that the calibration schedule is adhered to, detailed logs are maintained, and service labels are clearly displayed on equipment.

Use and cleaning advice for the designated molecular space

  • Pre-PCR: Reagent aliquoting / mastermix preparation

This should be the cleanest of all spaces used for the preparation of molecular experiments and should ideally be a designated laminar flow cabinet equipped with a UV light.
Samples, extracted nucleic acid, and amplified PCR products must not be handled in this area.
Amplification reagents should be kept in a freezer (or refrigerator, as per manufacturer recommendations) in the same designated space, ideally next to the laminar flow cabinet or pre-PCR area.
Gloves should be changed each time upon entering the pre-PCR area or laminar flow cabinet.
The pre-PCR area or laminar flow cabinet should be cleaned before and after use as follows: Wipe down all items in the cabinet, e.g. pipettes, tip boxes, vortex, centrifuge, tube racks, pens, etc. with 70% ethanol or a commercial DNA-destroying decontaminant, and allow to dry. In the case of a closed working area, e.g. a laminar flow cabinet, expose the hood to UV light for 30 minutes.

  • Pre-PCR: Nucleic acid extraction/template addition


Nucleic acid must be extracted and handled in a second designated area, using a separate set of pipettes, filter tips, tube racks, fresh gloves, lab coats, and other equipment.
This area is also for the addition of template, controls, and trendlines to the master mix tubes or plates. To avoid contamination of the extracted nucleic acid samples that are being analyzed, it is recommended to change gloves prior to handling positive controls or standards and to use a separate set of pipettes.
PCR reagents and amplified products must not be pipetted in this area.
Samples should be stored in designated fridges or freezers in the same area.
The sample workspace should be cleaned in the same way as the master mix space.

  • Post-PCR: Amplification and handling of the amplified product

This designated space is for post-amplification processes and should be physically separate from the pre-PCR areas. It usually contains thermocyclers and real-time platforms, and ideally should have a laminar flow cabinet for adding the round 1 PCR product to the round 2 reaction, if nested PCR is being performed.
PCR reagents and extracted nucleic acid must not be handled in this area since the risk of contamination are high.
This area should have a separate set of gloves, lab coats, plate and tube racks, pipettes, filter tips, bins, and other equipment.
Tubes must be centrifuged before opening.
The sample workspace should be cleaned in the same way as the master mix space.

Materials
MATERIALS
ReagentNuclease-Free Water, 150mlPromegaCatalog #P1195
ReagentLuna Universal Probe One-Step RT-qPCR Kit - 2,500 rxnsNew England BiolabsCatalog #E3006E
Reagent isopropyl alcoholSigmaCatalog #W292907
Reagent2-MercaptoethanolSigma AldrichCatalog #M3148
ReagentProteinase KNew England BiolabsCatalog #P8107S
Reagent200 Proof Ethanol pureSigma AldrichCatalog #E7023
ReagentRandom Hexamer PrimerThermo FisherCatalog #SO142
ReagentNEBNext Q5U Master Mix – 50 rxnsNew England BiolabsCatalog #M0597S
ReagentMagnesium Chloride Solution 1 M Sigma AldrichCatalog #M1028
ReagentsparQ PureMag BeadsQuantabioCatalog #95196-060
ReagentNextera DNA Flex Library Prep KitIllumina, Inc.
ReagentNN-DimethylformamideSigma AldrichCatalog #227056-1L
ReagentTRIS 1M pH 8.0VWR ScientificCatalog #E199-500mL
ReagentCorning 10% SDS (Sodium Dodecyl Sulfate)Fisher ScientificCatalog #MT-46040CI
ReagentIDT for Illumina DNA/RNA UD IndexesilluminaCatalog #20027213
ReagentQuick-DNA/RNA Viral MagbeadZymo ResearchCatalog #R2141
ReagentNN-DimethylformamideSigma AldrichCatalog #227056-100ML
ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G
ReagentNaCl - 1KgSigma AldrichCatalog #S3014-1KG
STEP MATERIALS
Reagent200 Proof Ethanol pureSigma AldrichCatalog #E7023
ReagentMineral OilMillipore SigmaCatalog #M5904
ReagentCorning 10% SDS (Sodium Dodecyl Sulfate)Fisher ScientificCatalog #MT-46040CI
ReagentQuick-DNA/RNA Viral MagbeadZymo ResearchCatalog #R2141
ReagentNextera DNA Flex Library Prep KitIllumina, Inc.
ReagentNN-DimethylformamideSigma AldrichCatalog #227056-100ML
ReagentMagnesium Chloride Solution 1 M Sigma AldrichCatalog #M1028
ReagentPoly(ethylene glycol) 8000Sigma Aldrich
ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G
ReagentTRIS 1M pH 8.0VWR ScientificCatalog #E199-500mL
ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G
ReagentTRIS 1M pH 8.0VWR ScientificCatalog #E199-500mL
ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G
ReagentPoly Ethylene Glycol (PEG) 8000Sigma AldrichCatalog #89510-250G-F
ReagentNaCl - 1KgSigma AldrichCatalog #S3014-1KG
ReagentQuick-DNA/RNA Viral MagbeadZymo ResearchCatalog #R2141
ReagentNuclease-Free Water, 150mlPromegaCatalog #P1195
ReagentTRIS 1M pH 8.0VWR ScientificCatalog #E199-500mL
ReagentLuna Universal Probe One-Step RT-qPCR Kit - 2,500 rxnsNew England BiolabsCatalog #E3006E
ReagentRandom HexamerThermo Fisher ScientificCatalog ##SO142
ReagentsparQ PureMag BeadsQuantabioCatalog #95196-060
ReagentIDT for Illumina DNA/RNA UD IndexesilluminaCatalog #20027213
ReagentNEBNext Q5U Master Mix – 50 rxnsNew England BiolabsCatalog #M0597S
ReagentsparQ PureMag BeadsQuantabioCatalog #95196-060
ReagentQuick-DNA/RNA Viral MagBeadZymo ResearchCatalog #R2141
ReagentBio-rad Hard-shell low-profile 96 well skirted PCR platesCatalog #HSP9601
ReagentProteinase KNew England BiolabsCatalog #P8107S
ReagentQuick-DNA/RNA Viral MagBeadZymo ResearchCatalog #R2141
Reagent2-MercaptoethanolSigma AldrichCatalog #M3148
Protocol materials
ReagentProteinase KNew England BiolabsCatalog #P8107S
In Materials, Materials, Step 3
ReagentBio-rad Hard-shell low-profile 96 well skirted PCR platesCatalog #HSP9601
Materials, Step 2
Reagent2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148
Materials, Step 5
Reagent2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148
Materials
ReagentNN-DimethylformamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #227056-100ML
In Materials, Materials, Step 28
ReagentTRIS 1M pH 8.0VWR InternationalCatalog #E199-500mL
In Materials, Materials, Materials, Materials and 3 steps
ReagentCorning 10% SDS (Sodium Dodecyl Sulfate)Fisher ScientificCatalog #MT-46040CI
In Materials, Materials, Step 30
ReagentRandom Hexamer PrimerThermo FisherCatalog #SO142
Materials
ReagentMagnesium Chloride Solution 1 M Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
In Materials, Materials, Step 28
ReagentNaCl - 1KgMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3014-1KG
In Materials, Materials, Step 35
ReagentQuick-DNA/RNA Viral MagbeadZymo ResearchCatalog #R2141
In Materials, Materials, Materials and 2 steps
ReagentQuick-DNA/RNA Viral MagBeadZymo ResearchCatalog #R2141
In Materials, Materials and 2 steps
ReagentPoly(ethylene glycol) 8000Merck MilliporeSigma (Sigma-Aldrich)
Materials, Step 33
ReagentNextera DNA Flex Library Prep KitIllumina, Inc.
In Materials, Materials, Step 28
Reagent200 Proof Ethanol pureMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
In Materials, Materials, Step 11
ReagentMineral OilMerck MilliporeSigma (Sigma-Aldrich)Catalog #M5904
Materials, Step 17
ReagentPoly Ethylene Glycol (PEG) 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510-250G-F
Materials, Step 35
Reagent isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907
Materials
ReagentNN-DimethylformamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #227056-1L
Materials
ReagentNEBNext Q5U Master Mix – 50 rxnsNew England BiolabsCatalog #M0597S
In Materials, Materials, Step 37
ReagentRandom HexamerThermo Fisher ScientificCatalog ##SO142
Materials, Step 16
ReagentNuclease-Free Water, 150mlPromegaCatalog #P1195
In Materials, Materials, Step 14
ReagentLuna Universal Probe One-Step RT-qPCR Kit - 2,500 rxnsNew England BiolabsCatalog #E3006E
In Materials, Materials, Step 16
ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G
In Materials, Materials, Materials, Materials and 3 steps
ReagentIDT for Illumina DNA/RNA UD IndexesIllumina, Inc.Catalog #20027213
In Materials, Materials, Step 37
ReagentsparQ PureMag BeadsQuantabioCatalog #95196-060
In Materials, Materials, Materials and 2 steps
Before start
  • Add 500 μl beta-mercaptoethanol per 100 ml Viral DNA/RNA Buffer (final concentrationof 0.5% (v/v)) from the Quick-DNA/RNA Viral MagBead.
  • Add 80 ml (R2141) of isopropanol to the MagBead DNA/RNA Wash 1 concentrate from the Quick-DNA/RNA Viral MagBead.
  • Add 120 ml (R2141) of isopropanol to the MagBead DNA/RNA Wash 2 concentrate from the Quick-DNA/RNA Viral MagBead.
RNA Extraction
RNA Extraction
1h
1h
For each saliva sample recieved add equal volume saliva and 2X DNA/RNA Shield from the Quick-DNA/RNA Viral magbead kit and vortex. Centrifuge the samples at Centrifigation500 rpm, 00:05:00 to bring down debris.

ReagentQuick-DNA/RNA Viral MagBeadZymo ResearchCatalog #R2141

Without disturbing the pellet, transfer Amount25 µL of saliva sample and 1X DNA/RNA to the bottom of a well in a 96-well deep-plate of each sample to a new tube
ReagentQuick-DNA/RNA Viral MagBeadZymo ResearchCatalog #R2141

ReagentBio-rad Hard-shell low-profile 96 well skirted PCR platesCatalog #HSP9601

Add Amount25 µL 1X DNA/RNA Shield supplemented with Amount2.5 µL of Proteinase K (20mg/mL) to each sample.
Briefly mix by using a plate shaker at Centrifigation1300 rpm, 25°C for Duration00:00:10 and incubate Duration00:15:00 at TemperatureRoom temperature .
Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK

ReagentProteinase KNew England BiolabsCatalog #P8107S

Remove Amount5 µL of MagBinding Beads per sample of the stock and place on a magnet stand. Incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completly clea. Then while avoiding the bead pellet, carefully remove the clear supernatnat. Resuspend the beads in Amount100 µL of Viral DNA/RNA Buffer (2-Mercaptoethanol 0.5% (v/v)) per sample and vortex to fully resuspend
Safety information
2-Mercaptoethanol is toxic, causing irritation to the nasal passageways and respiratory tract upon inhalation, irritation to the skin, vomiting and stomach pain through ingestion, and potentially death if severe exposure occurs.


Add Amount100 µL of the Viral DNA/RNA Buffer (2-Mercaptoethanol 0.5% (v/v)) and MagBinding Beads misture to each Amount50 µL sample in 1X DNA/RNA Shield. Mix by using a plate shaker at Centrifigation1300 rpm, 25°C for Duration00:10:00 .
Safety information
2-Mercaptoethanol is toxic, causing irritation to the nasal passageways and respiratory tract upon inhalation, irritation to the skin, vomiting and stomach pain through ingestion, and potentially death if severe exposure occurs.

Reagent2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148

Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment
SPRIPlate 96R Ring Super Magnet Plate
NAME
96-well Magnet Plate
TYPE
Agencourt
BRAND
A32782
SKU
https://www.beckman.com/supplies/plates/a32782
LINK

1m
Pipetting
Toxic
Add Amount100 µL MagBead DNA/RNA Wash 1 and mix by using a plate shaker at Centrifigation1300 rpm, 25°C for Duration00:02:00 .

ReagentQuick-DNA/RNA Viral MagbeadZymo ResearchCatalog #R2141

10m
Pipetting
Mix
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

1m
Pipetting
Add Amount100 µL MagBead DNA/RNA Wash 2 and mix by using a plate shaker at Centrifigation1300 rpm, 25°C for Duration00:02:00 .

ReagentQuick-DNA/RNA Viral MagbeadZymo ResearchCatalog #R2141


Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK

10m
Pipetting
Mix
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment
SPRIPlate 96R Ring Super Magnet Plate
NAME
96-well Magnet Plate
TYPE
Agencourt
BRAND
A32782
SKU
https://www.beckman.com/supplies/plates/a32782
LINK

1m
Pipetting
Add Amount150 µL 95-100% ethanol and mix by using a plate shaker at Centrifigation1300 rpm, 25°C for Duration00:02:00 .
Reagent200 Proof Ethanol pureMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023

10m
Pipetting
Mix
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment
SPRIPlate 96R Ring Super Magnet Plate
NAME
96-well Magnet Plate
TYPE
Agencourt
BRAND
A32782
SKU
https://www.beckman.com/supplies/plates/a32782
LINK

1m
Pipetting
Go to and repeat once.

To elute DNA/RNA from the beads, add Amount9 µL DNase/RNase-Free Water and mix using a plate shaker at
Centrifigation1300 rpm, 25°C for Duration00:01:00
ReagentNuclease-Free Water, 150mlPromegaCatalog #P1195

Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK

5m
Pipetting
Mix
Pellet the beads and transfer Amount7 µL of supernatant into a new tube. The eluted DNA/RNA can be used immediately or stored frozen at Temperature-80 °C .

Pause
RT-PCR
RT-PCR
2h
2h
Combine the following components into a thin-walled PCR tube.

Amount10 µL Luna Universal Probe One-Step Reaction Mix (2X)
Amount1 µL Luna WarmStart RT Enzyme Mix (20X)
Amount1 µL hCoV-2019/nCoV-2019 (V3) Primer Set mix (Primer pool 1 & 2) (100µm)
Amount1 µL Random Hexamers (100 µM)
Amount7 µL Viral RNA/DNA extract
ReagentLuna Universal Probe One-Step RT-qPCR Kit - 2,500 rxnsNew England BiolabsCatalog #E3006E

ReagentRandom HexamerThermo Fisher ScientificCatalog ##SO142

1m
Pipetting
Mix
Add Amount20 µL of mineral oil to each RT-PCR reaction to avoid evaporation and subsequent reaction failure.
ReagentMineral OilMerck MilliporeSigma (Sigma-Aldrich)Catalog #M5904

Pipetting
Set up and run the following RT-PCR program.

1. Reverse transcription: Duration00:10:00 at Temperature55 °C
2. Initial PCR activation: Duration00:01:00 at Temperature95 °C
3. 2-step PCR cycling (25X):
Denaturation: Duration00:00:15 at Temperature95 °C
Annealing & Extension: Duration00:03:00 at Temperature63 °C
4. Hold: Temperature4 °C

Equipment
Veriti 96-Well Thermal Cycler
NAME
Applied Biosystems
BRAND
4375786
SKU
https://www.thermofisher.com/order/catalog/product/4375786#/4375786
LINK

1h 30m
PCR
Add 0.7x volume of sparQ PureMag beads to each sample well in RT-PCR plate and mix gently by either flicking or pipetting. For example, add Amount14 µL sparQ PureMag beads to a Amount20 µL reaction. Note that the volume of oil is not taken into consideration given the oil is effectively inert. Then pulse centrifuge to collect all liquid at the bottom of the tube.

ReagentsparQ PureMag BeadsQuantabioCatalog #95196-060

1m
Pipetting
Mix
Incubate for Duration00:05:00 at TemperatureRoom temperature .

5m
Incubation
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment
SPRIPlate 96R Ring Super Magnet Plate
NAME
96-well Magnet Plate
TYPE
Agencourt
BRAND
A32782
SKU
https://www.beckman.com/supplies/plates/a32782
LINK


2m
Incubation
Keeping on the magnetic plate, add Amount150 µL of TemperatureRoom temperature freshly made Concentration80 % (v/v) ethanol to the side of the wall opposite to the pellet and let sit for Duration00:00:30 .

30s
Pipetting
Wash
Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for Duration00:00:10 then remove any remaining ethanol.

10s
Pipetting
Go to and repeat once.

Add 11 µl of Concentration10 mM Tris-HCl (Ph 8.0) and pipette to mix well. Incubate for Duration00:01:00 at Temperature37 °C .

ReagentTRIS 1M pH 8.0VWR InternationalCatalog #E199-500mL

Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK

1m
Incubation
Pipetting
Mix
Separate beads on the Agencourt SPRIPlate Super Magnet Plate for Duration00:02:00 or until the beads have pelleted.
Equipment
SPRIPlate 96R Ring Super Magnet Plate
NAME
96-well Magnet Plate
TYPE
Agencourt
BRAND
A32782
SKU
https://www.beckman.com/supplies/plates/a32782
LINK

2m
Incubation
Pellet the beads and transfer 10 µl of supernatant containing SARS-CoV-2 amplicons into a new tube. The eluted DNA can be used immediately or stored frozen at Temperature-20 °C .
Pipetting
Pause
Hackflex Library Preparation
Hackflex Library Preparation
2h
2h
Combine the following components into a thin-walled PCR tube.
Amount4 µL 5X Hacklfex Buffer (20mM Tris, 20 mM MgCl, 50% DMF)
Amount5.5 µL Nuclease-Free Water
Amount0.5 µL Enrichment Bead-Linked Transposomes (eBLT)
Amount10 µL SARS-CoV-2 amplicons

ReagentNextera DNA Flex Library Prep KitIllumina, Inc.

ReagentNN-DimethylformamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #227056-100ML

ReagentMagnesium Chloride Solution 1 M Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028

Pipetting
Mix
Set up and run the following thermocycler program.

1. Tagmentation: Duration00:05:00 at Temperature55 °C
2. Hold: Temperature10 °C

Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK

15m
Incubation
Add Amount5 µL of Hackflex Stop Buffer (0.2% SDS) to the sample.

ReagentCorning 10% SDS (Sodium Dodecyl Sulfate)Fisher ScientificCatalog #MT-46040CI

1m
Pipetting
Set up and run the following thermocycler program.

1. Tagmentation: Duration00:05:00 at Temperature25 °C
2. Hold: Temperature10 °C

Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK

15m
Incubation
Place the plates on the Agencourt SPRIPlate Super Magnet Plate and incubate for Duration00:05:00 or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment
SPRIPlate 96R Ring Super Magnet Plate
NAME
96-well Magnet Plate
TYPE
Agencourt
BRAND
A32782
SKU
https://www.beckman.com/supplies/plates/a32782
LINK

Incubation
Pipetting
Keeping on the magnetic plate, add Amount30 µL of TemperatureRoom temperature Hackflex Wash Buffer (10% PEG 8000, 0.25 M NaCl, 10mM Tris-HCL pH 8.0, 0.1mM EDTA) gently mix and then let sit for Duration00:00:30 .

ReagentPoly(ethylene glycol) 8000Merck MilliporeSigma (Sigma-Aldrich)

ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G

30s
Incubation
Pipetting
Mix
Separate beads on the Agencourt SPRIPlate Super Magnet Plate for Duration00:02:00 or until the beads have pelleted.

Equipment
SPRIPlate 96R Ring Super Magnet Plate
NAME
96-well Magnet Plate
TYPE
Agencourt
BRAND
A32782
SKU
https://www.beckman.com/supplies/plates/a32782
LINK

2m
Avoid disturbing the bead pellet, carefully remove and discard HWB (10% PEG 8000, 0.25 M NaCl, 10mM Tris-HCL pH 8.0, 0.1mM EDTA).

ReagentPoly Ethylene Glycol (PEG) 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510-250G-F


ReagentNaCl - 1KgMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3014-1KG

ReagentTRIS 1M pH 8.0VWR InternationalCatalog #E199-500mL

ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G

30s
Pipetting
Go to and repeat once.
Resuspend beads in the following Amount20 µL Indexing-PCR Master Mix

Amount10 µL NEBNext Q5U Master Mix (2X)
Amount1 µL IDT for Illumina DNA/RNA UD Indexes
Amount9 µL Nuclease-free Water

ReagentNEBNext Q5U Master Mix – 50 rxnsNew England BiolabsCatalog #M0597S

ReagentIDT for Illumina DNA/RNA UD IndexesIllumina, Inc.Catalog #20027213

1m
Pipetting
Mix
Set up and run the following Indexing-PCR program.

1. Initial Denaturation: Duration00:01:00 at Temperature98 °C
2. 3-step PCR cycling (6X):
Denaturation: Duration00:00:15 at Temperature98 °C
Annealing: Duration00:00:30 at Temperature62 °C
Extension: Duration00:00:30 at Temperature65 °C
3. Final Extension: Duration00:01:00 at Temperature65 °C
4. Hold: Temperature4 °C

Equipment
Veriti 96-Well Thermal Cycler
NAME
Applied Biosystems
BRAND
4375786
SKU
https://www.thermofisher.com/order/catalog/product/4375786#/4375786
LINK

20m
PCR
After PCR, pool Amount10 µL of each sample into a single tube and vortex to mix.

Pipetting
Add an equal volume (1:1) of sparQ PureMag beads to the library pool and mix gently by either flicking or pipetting. For example, add Amount25 µL sparQ PureMag beads to a Amount25 µL reaction. Then pulse centrifuge to collect all liquid at the bottom of the tube.

ReagentsparQ PureMag BeadsQuantabioCatalog #95196-060

1m
Pipetting
Mix
Incubate for Duration00:05:00 at TemperatureRoom temperature .

5m
Incubation
Place the pool on a magnet and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.


Equipment
Magnetic Stand
NAME
Magnetic Stand
TYPE
Thermo Scientific
BRAND
MR02
SKU
https://www.thermofisher.com/order/catalog/product/MR02
LINK
Any magnetic rack that fits your tubes will suffice.
SPECIFICATIONS


2m
Keeping the pool on the magnet, add Amount200 µL of TemperatureRoom temperature freshly made Concentration80 % (v/v) ethanol to the side of the wall opposite to the pellet and let sit for Duration00:00:30 .

30s
Incubation
Pipetting
Wash
Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for Duration00:00:10 then remove any remaining ethanol.

10s
Pipetting
Go to and repeat once.

Add 25 µl of Concentration10 mM TE Buffer (10mM Tris-HCL pH 8.0, 0.1mM EDTA) and pipette to mix well. Incubate for Duration00:01:00 at Temperature37 °C .

ReagentTRIS 1M pH 8.0VWR InternationalCatalog #E199-500mL

ReagentEDTA 100mLThermo Fisher ScientificCatalog #AM9260G

1m
Incubation
Pipetting
Quantifying Pool and Sequencing
Quantifying Pool and Sequencing
Quantify final pool with adapted libraries. Load onto sequencer using platform appropriate dilutions.


Equipment
MiSeq
NAME
Sequencer
TYPE
illumina
BRAND
SY-410-1003
SKU
https://www.illumina.com/systems/sequencing-platforms/miseq/order-miseq.html
LINK