TITLE: Pathogen-Oriented Low-cost Assembly & Re-sequencing (POLAR): A highly sensitive and high-throughput SARS-CoV-2 diagnostic based on whole genome sequencing
AUTHORS: Brian Glenn St Hilaire, 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, Aviva Presser Aiden, Erez Lieberman Aiden

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>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 </span><span style = "font-style:italic;">de novo </span><span>genome assembly. For details on our computational pipeline for automated data processing, including documentation and test set, please visit our Githuh at </span><a href="https://github.com/aidenlab/Polar" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/aidenlab/Polar</span></a><span style = ":;">.</span></div><style>
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[STEPS]
?. [cDNA preparation]
Make a mastermix of the dNTPs and Random Hexamers for 96 samples (account for pipette error), pipette to mix and add  from mastermix to each well in a 96-well plate. To each well, add  of RNA extract eluted in Step 7.
1 µl
5.5 µl
?. [cDNA preparation]
Set-up and run the following program on a thermal cycler to incubate reaction:
?. [cDNA preparation]
Make a mastermix of the following components for 96 samples (account for pipette error) and mix by pipetting.Add  of mastermix to each well with the annealed template RNA from Step 9.
3.5 µl
?. [cDNA preparation]
Set-up and run the following program on a thermal cycler to incubate reaction:
?. [Multiplex PCR]
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  of the corresponding Pool master mix into each well.
22.5 µl
?. [Multiplex PCR]
Set-up and run the following program on a thermal cycler to incubate reaction
?. [Post PCR Clean-up]
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  sparQ PureMag beads to a   reaction. Then pulse centrifuge to collect all liquid at the bottom of the tube.
50 µl
50 µl
?. [Post PCR Clean-up]
Incubate for   at room temperature.
?. [Post PCR Clean-up]
Place the plates on the Agencourt SPRIPlate Super Magnet Plate and incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [Post PCR Clean-up]
Keeping on magnetic plate, add  of  ethanol to the side of the well opposite to the pellet and let sit for .
200 µl
0 Room temperature
[(freshly made)]
?. [Post PCR Clean-up]
Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for   then remove any remaining ethanol.
?. [Post PCR Clean-up]
Add of  Tris-HCl (Ph 8.0) and pipette to mix well. Incubate for  at  .
20 µl
37 °C
?. [Post PCR Clean-up]
and repeat ethanol wash.
?. [Nextera Library Preparation]
Make a master mix of the Tagment mix and Tagment Buffer for 96 samples (account for pipette error) and pipette to mix well. Aliquot  of the master mix into each well of a new 96-well plate and add  of 1ng Amplicon DNA.
6 µl
4 µl
?. [Nextera Library Preparation]
Incubate at  for, then lower to . Once at 10°C, immediately add  of Neutralizing Tagment Buffer to stop the reaction. Mix by pipetting up and down.
55 °C
10 °C
2.5 µl
?. [Nextera Library Preparation]
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  of this mastermix into each well of a new 96-well plate and add  of tagmented amplicon DNA from Step 35.
12.5 µl
12.5 µl
?. [Nextera Library Preparation]
Set-up and run the following program on a thermal cycler to incubate reaction:
?. [Final Library Cleanup]
Incubate for   at room temperature.
?. [Final Library Cleanup]
Place the plate on the Agencourt SPRIPlate Super Magnet Plate and incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [Final Library Cleanup]
Add a 1:1.8 ratio of sparQ PureMag beads to each well and mix gently pipetting. For example, add  sparQ PureMag beads to a  reaction.
90 µl
50 µl
?. [Final Library Cleanup]
While keeping on magnetic plate, add  of  ethanol to the side of the well opposite of the pellet and let sit for .
200 µl
0 Room temperature
[(fresh made)]
?. [Final Library Cleanup]
Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for   then remove any remaining ethanol.
?. [Final Library Cleanup]
Add of  Tris-HCl (pH 8.0) , vortex, and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for  at .
100 µl
37 °C
?. [Final Library Cleanup]
Add an equal volume (1:1) of sparQ PureMag beads to each well and mix gently by pipetting. For example add  sparQ PureMag beads to a   reaction.
100 µl
100 µl
?. [Final Library Cleanup]
Incubate for   at room temperature.
?. [Final Library Cleanup]
Separate beads on the Agencourt SPRIPlate Super Magnet Plate and incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [Final Library Cleanup]
While keeping on magnetic plate, add  of  ethanol to the side of the well opposite of the pellet and let sit for .
200 µl
0 Room temperature
[(fresh made)]
?. [Final Library Cleanup]
Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for   then remove any remaining ethanol.
?. [Final Library Cleanup]
and repeat ethanol wash.
?. [Final Library Cleanup]
Add of  Tris-HCl (Ph 8.0) and mix well by pipetting. Incubate for  at
20 µl
37 °C
?. [Final Library Cleanup]
Place the plate on the Agencourt SPRIPlate Super Magnet Plate and incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, transfer the  final library to a new plate for subsequent steps and storage.
20 µl
?. [cDNA preparation]
?. [RNA extraction]
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   i.e.,   per 100 ml.2. Add   of 100% ethanol (or  of 95% ethanol) to the  Viral Wash Buffer concentrate.
500 µl
192 ml
204 ml
48 ml
?. [RNA extraction]
Mount the Zymo-Spin™ I-96 plate on a Collection Plate and centrifuge at - for . Discard the flow-through from the collection plate.
Centrifuge: 3000 34
Centrifuge: 5000 34
?. [RNA extraction]
Add Viral Wash Buffer to each well and centrifuge at - for . Discard the flowthrough from the collection plate. Repeat this step.
500 µl
Centrifuge: 3000 34
Centrifuge: 5000 34
?. [RNA extraction]
Add  ethanol (95-100%) to each well and centrifuge at  -  for .
500 µl
Centrifuge: 3000 34
Centrifuge: 5000 34
?. [RNA extraction]
To elute RNA, mount a new 96-well plate between the Zymo-Spin™ I-96 plate and an empty Collection Plate. Add   DNase/RNase-Free Water directly to the column matrix of the Zymo-Spin™ I-96 plate wells and centrifuge at  -  for . The eluted RNA extract will be collected in the middle 96-well plate.
15 µl
Centrifuge: 3000 34
Centrifuge: 5000 34
?. [RNA extraction]
Add  of Viral RNA Buffer to each sample well. Mix well by pipetting.
400 µl
?. [Loading the NextSeq550 sequencer]
Remove a “Mid-Output Kit" reagent cartridge from -20°C freezer storage.
?. [Loading the NextSeq550 sequencer]
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  .
?. [Loading the NextSeq550 sequencer]
When the reagent cartridge has been thawing for , 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   to warm up to
0 Room temperature
?. [Loading the NextSeq550 sequencer]
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.
?. [Loading the NextSeq550 sequencer]
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.
?. [Loading the NextSeq550 sequencer]
Remove the flow cell from its packaging and gently clean with a 70% ethanol wipe. Dry the glass with another wipe.
?. [Loading the NextSeq550 sequencer]
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.
?. [Loading the NextSeq550 sequencer]
Remove a tube of Hybridization Buffer (HT1) from the -20°C freezer and thaw at  . When thawed, place on ice.
0 Room temperature
?. [Loading the NextSeq550 sequencer]
In a fresh 1.5ml tube, combine an aliquot from each library pool such that the total volume is . For a Mid-Output kit, expect to obtain 150M reads in total.
5 µl
?. [Loading the NextSeq550 sequencer]
Add  of ice-cold Hybridization Buffer (HT1). Vortex briefly and centrifuge at  for .
995 µl
Centrifuge: 300 34
?. [Loading the NextSeq550 sequencer]
In a fresh 1.5ml tube, combine  of ice-cold Hybridization Buffer (HT1) and  diluted library for a final concentration of . Invert to mix and pulse centrifuge. Place on ice until ready to load onto the reagent cartridge.
1203 µl
97 µl
?. [Loading the NextSeq550 sequencer]
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:
?. [Loading the NextSeq550 sequencer]
Add the entire 1.3ml of the  library dilution into this reservoir.
?. [Loading the NextSeq550 sequencer]
The reagent cartridge, flow cell, and buffer pack are now ready to be loaded onto the NextSeq500 for sequencing.
?. [Library Normalization ]
Transfer  supernatant from each well of the PCR plate to the corresponding well of a new midi plate.
10 µl
?. [Library Normalization ]
Combine the following volumes in a  conical tube to prepare the LN master mix. Multiply each volume by the number of samples being processed:   of LNA1 and  of LNA2.
15 ml
23 µl
4 µl
?. [Library Normalization ]
Pipette 10 times to mix and pour the LN master mix into a trough.
?. [Library Normalization ]
Use a p200 multichannel pipette to transfer  LN master mix to each sample well.
22.5 µl
?. [Library Normalization ]
Seal the plate, and then use a plate shaker at  for
Centrifuge: 1800 33
?. [Library Normalization ]
Place on the magnetic stand and wait until the liquid is clear (~2 minutes). Without disturbing the beads, discard all the supernatant.
?. [Library Normalization ]
Wash two times as follows: Add  LNW1 to each well. Seal the plate, and then use a plate shaker at  for. Separate on a magnetic plate and wait until the liquid is clear (~2 minutes). Without disturbing the beads, remove and discard all supernatant.
22.5 µl
Centrifuge: 1800 33
?. [Library Normalization ]
Add  0.1 N NaOH to each well. Seal the plate, and then use a plate shaker at  for
15 µl
Centrifuge: 1800 33
?. [Library Normalization ]
Add  LNS1 to each well of a new 96-well PCR plate labeled SGP.
15 µl
?. [Library Normalization ]
After the  elution completes, make sure that all samples in the midi plate are resuspended. If they are not, resuspend as follows:
?. [Library Normalization ]
Pipette 10 times to mix or lightly tap the sample plate on the bench. Seal the plate, and then use a plate shaker at  for . Place on a magnet rack and transfer  supernatant from each well of the midi plate to thecorresponding well of the SGP plate.
Centrifuge: 1800 33
15 µl
?. [Library Normalization ]
Seal the plate, and then centrifuge at  for.
Centrifuge: 1000 34
?. [RNA extraction]
Collect 96 patient samples and record sample data. Add  of the samples into each well of the Zymo-Spin™ I-96 plate. Keep plate  while preparing reagents in Step 2.
200 µl
on ice
?. [Multiplex PCR]
Add  of cDNA of each biological sample from from Step 11 to both Pool #1 and Pool #2 96-well plates.
2.5 µl
?. [Post PCR Clean-up]
Separate beads on the Agencourt SPRIPlate Super Magnet Plate for  or until the beads have pelleted.
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Quantify the DNA concentration using the Qubit® High Sensitivity DNA kit from   of each product using Steps 26-35.
1 µl
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Prepare a working solution by diluting the Qubit® dsDNA HS Reagent 1:200 in Qubit® dsDNA HS Buffer. For example, add  of buffer and  of dye per sample to a tube and vortex.
199 µl
1 µl
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Add  of Qubit® working solution and  of the standard to each of the standards tubes and vortex.
190 µl
10 µl
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
For each sample, add  of Qubit® working solution and  of the sample in each 0.5-mL sample tube and vortex.
199 µl
1 µl
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Allow all tubes to incubate at room temperature for
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
On the Home screen of the Qubit® 3.0 Fluorometer, press DNA, select dsDNA High Sensitivity, and press Read Standards to proceed.
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Read standard. When the reading is complete (~), remove Standard #1.
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Repeat Step 32 with Standard #2.
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
To quantify samples, Press Run samples and select the sample volume ( ) and units.
1 µl
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Repeat Step 34 for all samples.
?. [Post PCR Clean-up]
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.
?. [Final Library Cleanup]
After PCR, bring up sample volume in each well to  with nuclease free water.
50 µl
?. [Final Library Cleanup]
Separate beads on the Agencourt SPRIPlate Super Magnet Plate and transfer  of supernatant to a new plate for a second round of cleanup.
100 µl
?. [Multiplex PCR]
The following steps are performed in plates prepared according to the schematic below:
?. [Loading the NextSeq550 sequencer]
Preheat an incubator to
98 °C
?. [Loading the NextSeq550 sequencer]
Transfer of diluted library to a new tube and add  of ice-cold Hybridization Buffer (HT1).
750 µl
750 µl
?. [Loading the NextSeq550 sequencer]
Vortex briefly and centrifuge at  for .
Centrifuge: 300 34
?. [Loading the NextSeq550 sequencer]
Place the tube on the  heated incubator for  and immediately cool on ice. Leave on ice for
98 °C
?. [Quantification of DNA concentration using a Qubit® High Sensitivity Kit:]
Set up the required number of 0.5-mL tubes for standards and samples and label lids accordingly.
?. [Nextera Library Preparation]
Incubate at   for . Centrifuge at  for .
10 Room temperature
Centrifuge: 280 34
?. [Library Normalization ]
Place on a magnetic stand and wait until the liquid is clear ~ .
?. [Library Normalization ]
Transfer  supernatant from each well of the midi plate to the corresponding well of the SGP plate.
15 µl
?. [Library Normalization ]
Seal the sample plate, and then centrifuge at for .
Centrifuge: 1000 34