TITLE: Pathogen-Oriented Low-cost Assembly &amp; Re-sequencing (POLAR): A highly sensitive and high-throughput SARS-CoV-2 diagnostic based on whole genome sequencing
AUTHORS: 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

[DESCRIPTION]
Here, we introduce a low-cost, high-throughput method for diagnosis of SARS-CoV-2 infection, dubbed Pathogen- Oriented Low-Cost Assembly & Re-Sequencing (POLAR), that enhances sensitivity by aiming to amplify the entire SARS-CoV-2 genome rather than targeting particular viral loci, as in typical RT- PCR assays. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. For details on our computational pipeline for automated data processing, including documentation and test set, please visit our Githuh at https://github.com/aidenlab/Polar.

The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.

[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.  

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:
master mix preparation,
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.


[STEPS]
SECTION: cDNA preparation
8. Make a mastermix of the dNTPs and Random Hexamers for 96 samples (account for pipette error), pipette to mix and add 1 µL from mastermix to each well in a 96-well plate. 

To each well, add 5.5 µL of RNA extract eluted in Step 7. 
 

SECTION: cDNA preparation
9.  
Set-up and run the following program on a thermal cycler to incubate reaction:

 
    


SECTION: cDNA preparation
10. Make a mastermix of the following components for 96 samples (account for pipette error) and mix by pipetting.
Add 3.5 µL of mastermix to each well with the annealed template RNA from Step 9. 


 

SECTION: cDNA preparation
11. Set-up and run the following program on a thermal cycler to incubate reaction:

 
 
  

SECTION: Multiplex PCR
13. Make a Pool #1 and Pool #2 master mix with the following reagents for 96 samples (account for pipette error) and mix by pipetting. In two 96-well plates, labeled Pool #1 or Pool #2, aliquot 22.5 µL of the corresponding Pool master mix into each well.


 
 

SECTION: Multiplex PCR
15. Set-up and run the following program on a thermal cycler to incubate reaction

 



SECTION: Post PCR Clean-up
16. Add an equal volume (1:1) of sparQ PureMag beads to each sample well in plates Pool #1 and Pool #2 and mix gently by either flicking or pipetting. For example, add 50 µL sparQ PureMag beads to a 50 µL  reaction. Then pulse centrifuge to collect all liquid at the bottom of the tube.


SECTION: Post PCR Clean-up
17. Incubate for 5 min  at room temperature.

SECTION: Post PCR Clean-up
18. Place the plates on the Agencourt SPRIPlate Super Magnet Plate and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.  

 


SECTION: Post PCR Clean-up
19. Keeping on magnetic plate, add 200 µL of Room temperature 80 % volume ethanol to the side of the well opposite to the pellet and let sit for 30 s. 


SECTION: Post PCR Clean-up
20. Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for  10 s then remove any remaining ethanol. 

SECTION: Post PCR Clean-up
22. Add 20 µLof 10 millimolar (mM) Tris-HCl (Ph 8.0) and pipette to mix well. Incubate for 2 min at 37 °C . 

SECTION: Post PCR Clean-up
21.    and repeat ethanol wash.
SECTION: Nextera Library Preparation
36. Make a master mix of the Tagment mix and Tagment Buffer for 96 samples (account for pipette error) and pipette to mix well. Aliquot 6 µL of the master mix into each well of a new 96-well plate and add 4 µL of 1ng Amplicon DNA.
 

SECTION: Nextera Library Preparation
37. Incubate at 55 °C for5 min, then lower to 10 °C. Once at 10°C, immediately add 2.5 µL of Neutralizing Tagment Buffer to stop the reaction. Mix by pipetting up and down.


SECTION: Nextera Library Preparation
39. Make a mastermix of the Nextera PCR Master Mix and Index Primers 1 and 2 for 96 samples (account for pipette error) and mix by pipetting.  Aliquot 12.5 µL of this mastermix into each well of a new 96-well plate and add 12.5 µL of tagmented amplicon DNA from Step 35. 

 
 
 



SECTION: Nextera Library Preparation
40. Set-up and run the following program on a thermal cycler to incubate reaction:

 
   

SECTION: Final Library Cleanup
43. Incubate for 5 min  at room temperature.

SECTION: Final Library Cleanup
44. Place the plate on the Agencourt SPRIPlate Super Magnet Plate and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.  




SECTION: Final Library Cleanup
42. Add a 1:1.8 ratio of sparQ PureMag beads to each well and mix gently pipetting. For example, add 90 µL sparQ PureMag beads to a 50 µL reaction. 

SECTION: Final Library Cleanup
45. While keeping on magnetic plate, add 200 µL of Room temperature 80 % volume ethanol to the side of the well opposite of the pellet and let sit for 30 s. 


SECTION: Final Library Cleanup
46. Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for  10 s then remove any remaining ethanol.

SECTION: Final Library Cleanup
47. Add 100 µLof 10 millimolar (mM) Tris-HCl (pH 8.0) , vortex, and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 2 min at 37 °C. 

SECTION: Final Library Cleanup
49. Add an equal volume (1:1) of sparQ PureMag beads to each well and mix gently by pipetting. For example add 100 µL sparQ PureMag beads to a 100 µL  reaction. 
SECTION: Final Library Cleanup
50. Incubate for 5 min  at room temperature.
SECTION: Final Library Cleanup
51. Separate beads on the Agencourt SPRIPlate Super Magnet Plate and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant. 

SECTION: Final Library Cleanup
52. While keeping on magnetic plate, add 200 µL of Room temperature 80 % volume ethanol to the side of the well opposite of the pellet and let sit for 30 s. 

SECTION: Final Library Cleanup
53. Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for  10 s then remove any remaining ethanol.

SECTION: Final Library Cleanup
54.    and repeat ethanol wash. 

SECTION: Final Library Cleanup
55. Add 20 µLof 10 millimolar (mM) Tris-HCl (Ph 8.0) and mix well by pipetting. Incubate for 2 min at 37 °C 

SECTION: Final Library Cleanup
56. Place the plate on the Agencourt SPRIPlate Super Magnet Plate and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, transfer the 20 µL final library to a new plate for subsequent steps and storage. 

SECTION: RNA extraction
2. Prepare the following reagents before starting viral RNA extraction:
1. Add beta-mercaptoethanol (user supplied) to the Viral RNA Buffer to a final dilution of 0.5 % volume  i.e., 500 µL  per 100 ml.
2. Add 192 mL  of 100% ethanol (or 204 mL of 95% ethanol) to the 48 mL Viral Wash Buffer concentrate.

SECTION: RNA extraction
4. Mount the Zymo-Spin™ I-96 plate on a Collection Plate and centrifuge at 3000 x g-5000 x g for 5 min. Discard the flow-through from the collection plate.

SECTION: RNA extraction
5. Add 500 µLViral Wash Buffer to each well and centrifuge at 3000 x g-5000 x g for 5 min. Discard the flowthrough from the collection plate. Repeat this step.

SECTION: RNA extraction
6. Add 500 µL ethanol (95-100%) to each well and centrifuge at 3000 x g - 5000 x g for 5 min. 

SECTION: RNA extraction
7. To elute RNA, mount a new 96-well plate between the Zymo-Spin™ I-96 plate and an empty Collection Plate. Add 15 µL  DNase/RNase-Free Water directly to the column matrix of the Zymo-Spin™ I-96 plate wells and centrifuge at 3000 x g - 5000 x g for 5 min. The eluted RNA extract will be collected in the middle 96-well plate.


SECTION: RNA extraction
3. Add 400 µL of Viral RNA Buffer to each sample well. Mix well by pipetting.
SECTION: Loading the NextSeq550 sequencer
70. Remove a “Mid-Output Kit" reagent cartridge from -20°C freezer storage.

 

SECTION: Loading the NextSeq550 sequencer
71. Place the cartridge right-side-up in a secondary container and fill the container with water at room temperature, but do not fully submerge the cartridge. Thaw cartridge in this water bath for about 60 min .

SECTION: Loading the NextSeq550 sequencer
72. When the reagent cartridge has been thawing for 30 min, remove a "Mid-Output Kit" flow cell from the 4°C refrigerator but do not open the foil package.  Place the flow cell on the bench for 30 min  to warm up to Room temperature  
 

SECTION: Loading the NextSeq550 sequencer
73. After the reagent cartridge is fully thawed, take it out of the water bath, tap on the bench to dislodge excess water, and dry with wipes.


SECTION: Loading the NextSeq550 sequencer
74. Invert the cartridge to mix the reagents and make sure that the sequencing reagents in positions 29-32 are completely thawed. Then tap on the bench again to reduce air bubbles.


SECTION: Loading the NextSeq550 sequencer
75. Remove the flow cell from its packaging and gently clean with a 70% ethanol wipe. Dry the glass with another wipe. 

SECTION: Loading the NextSeq550 sequencer
76. Inspect the flow cell to confirm: ports are free of obstructions, port gaskets are sealed and white plastic posts are visible, all 4 white retention clips are snapped over the edge of the black carrier plate and all 4 metal spring clips are laying flat against the black carrier plate.
SECTION: Loading the NextSeq550 sequencer
78. Remove a tube of Hybridization Buffer (HT1) from the -20°C freezer and thaw at Room temperature . When thawed, place on ice.

SECTION: Loading the NextSeq550 sequencer
79. In a fresh 1.5ml tube, combine an aliquot from each library pool such that the total volume is 5 µL. For a Mid-Output kit, expect to obtain 150M reads in total. 


SECTION: Loading the NextSeq550 sequencer
80. Add 995 µL of ice-cold Hybridization Buffer (HT1). Vortex briefly and centrifuge at 300 x g for 1 min. 

SECTION: Loading the NextSeq550 sequencer
84. In a fresh 1.5ml tube, combine 1203 µL of ice-cold Hybridization Buffer (HT1) and 97 µL diluted library for a final concentration of 1.5 picomolar (pM). Invert to mix and pulse centrifuge. Place on ice until ready to load onto the reagent cartridge.

SECTION: Loading the NextSeq550 sequencer
85. Part 3: Load the Sequencer 

Using a clean P1000 tip, poke a hole in the foil seal covering position 10 on the reagent cartridge (labeled “Load Library Here”). Avoid breaking any of the other seals on the cartridge:

 


SECTION: Loading the NextSeq550 sequencer
86. Add the entire 1.3ml of the 1.5 picomolar (pM) library dilution into this reservoir.

SECTION: Loading the NextSeq550 sequencer
87. The reagent cartridge, flow cell, and buffer pack are now ready to be loaded onto the NextSeq500 for sequencing. 
SECTION: Library Normalization
57. Transfer 10 µL supernatant from each well of the PCR plate to the corresponding well of a new midi plate.

SECTION: Library Normalization
58. Combine the following volumes in a 15 mL conical tube to prepare the LN master mix. Multiply each volume by the number of samples being processed:  23 µL of LNA1 and 4 µL of LNA2.

SECTION: Library Normalization
59. Pipette 10 times to mix and pour the LN master mix into a trough.
SECTION: Library Normalization
60. Use a p200 multichannel pipette to transfer 22.5 µL LN master mix to each sample well.

SECTION: Library Normalization
61. Seal the plate, and then use a plate shaker at 1800 rpm for 30 min

SECTION: Library Normalization
62. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). Without disturbing the beads, discard all the supernatant. 
SECTION: Library Normalization
63. Wash two times as follows: 
Add 22.5 µL LNW1 to each well. Seal the plate, and then use a plate shaker at 1800 rpm for5 min. Separate on a magnetic plate and wait until the liquid is clear (~2 minutes). Without disturbing the beads, remove and discard all supernatant.

SECTION: Library Normalization
64. Add 15 µL 0.1 N NaOH to each well. Seal the plate, and then use a plate shaker at 1800 rpm for 5 min

SECTION: Library Normalization
65. Add 15 µL LNS1 to each well of a new 96-well PCR plate labeled SGP.

SECTION: Library Normalization
66. After the 5 min elution completes, make sure that all samples in the midi plate are resuspended. If they are not, resuspend as follows: 

SECTION: Library Normalization
66.1. Pipette 10 times to mix or lightly tap the sample plate on the bench. Seal the plate, and then use a plate shaker at 1800 rpm for 5 min. Place on a magnet rack and transfer 15 µL supernatant from each well of the midi plate to thecorresponding well of the SGP plate. 

SECTION: Library Normalization
66.2. Seal the plate, and then centrifuge at 1000 x g for1 min.


SECTION: RNA extraction
1. Collect 96 patient samples and record sample data. Add 200 µL of the samples into each well of the Zymo-Spin™ I-96 plate. Keep plate on ice while preparing reagents in Step 2. 


SECTION: Multiplex PCR
14. Add 2.5 µL of cDNA of each biological sample from from Step 11 to both Pool #1 and Pool #2 96-well plates.

SECTION: Post PCR Clean-up
23. Separate beads on the Agencourt SPRIPlate Super Magnet Plate for 2 min or until the beads have pelleted. 

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
25. Quantify the DNA concentration using the Qubit® High Sensitivity DNA kit from 1 µL  of each product using Steps 26-35. 


SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
27. Prepare a working solution by diluting the Qubit® dsDNA HS Reagent 1:200 in Qubit® dsDNA HS Buffer. For example, add 199 µL of buffer and 1 µL of dye per sample to a tube and vortex. 

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
28. Add 190 µL of Qubit® working solution and 10 µL of the standard to each of the standards tubes and vortex.

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
29. For each sample, add 199 µL of Qubit® working solution and 1 µL of the sample in each 0.5-mL sample tube and vortex.

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
30. Allow all tubes to incubate at room temperature for 2 min

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
31. On the Home screen of the Qubit® 3.0 Fluorometer, press DNA, select dsDNA High Sensitivity, and press Read Standards to proceed.
SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
32. Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Read standard. When the reading is complete (~3 s), remove Standard #1.

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
33. Repeat Step 32 with Standard #2.
SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
34. To quantify samples, Press Run samples and select the sample volume (1 µL ) and units.

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
35. Repeat Step 34 for all samples.
SECTION: Post PCR Clean-up
24. Transfer supernatant from each biological sample in the two plates (Pool #1 and Pool #2) and combine into a single well per sample in a new 96-well plate. 
SECTION: Final Library Cleanup
41. After PCR, bring up sample volume in each well to 50 µL with nuclease free water.

SECTION: Final Library Cleanup
48.  Separate beads on the Agencourt SPRIPlate Super Magnet Plate and transfer 100 µL of supernatant to a new plate for a second round of cleanup. 


SECTION: Multiplex PCR
12. The following steps are performed in plates prepared according to the schematic below:

 

SECTION: Loading the NextSeq550 sequencer
77. Preheat an incubator to 98 °C 

SECTION: Loading the NextSeq550 sequencer
81. Transfer 750 µLof diluted library to a new tube and add 750 µL of ice-cold Hybridization Buffer (HT1).

SECTION: Loading the NextSeq550 sequencer
82. Vortex briefly and centrifuge at 300 x g for 1 min. 

SECTION: Loading the NextSeq550 sequencer
83. Place the tube on the 98 °C heated incubator for 2 min and immediately cool on ice. Leave on ice for 5 min

SECTION: Quantification of DNA concentration using a Qubit® High Sensitivity Kit:
26. Set up the required number of 0.5-mL tubes for standards and samples and label lids accordingly.
SECTION: Nextera Library Preparation
38. Incubate at  10 Room temperature for 5 min. Centrifuge at 280 x g for 1 min.

SECTION: Library Normalization
67. Place on a magnetic stand and wait until the liquid is clear ~ 2 min.

SECTION: Library Normalization
68. Transfer 15 µL supernatant from each well of the midi plate to the corresponding well of the SGP plate.

SECTION: Library Normalization
69. Seal the sample plate, and then centrifuge at1000 x g for 1 min.

SECTION: Spotlight video
88.