Nov 03, 2025

Public workspaceMetagenomic sequencing protocol for respiratory virus detection and sequencing from negative SARS-CoV-2 rapid antigen tests

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l29pqjv1y/v1
  • Emmanuela Jules1,
  • Alaa Ahmed2,
  • Hannah Dakanay1,
  • Anne Piantadosi MD, PhD1,3
  • 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, 30322, USA;
  • 2Emory University;
  • 3Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA, 30322, USA
  • Anne Piantadosi MD, PhD: Corresponding author;
Hannah Cayla C. Dakanay
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l29pqjv1y/v1
Protocol Citation: Emmanuela Jules, Alaa Ahmed, Hannah Dakanay, Anne Piantadosi MD, PhD 2025. Metagenomic sequencing protocol for respiratory virus detection and sequencing from negative SARS-CoV-2 rapid antigen tests. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l29pqjv1y/v1
Manuscript citation:
Jules, E., Decker, C., Bixler, B. J., Ahmed, A., Zhou, Z. C., Arora, I., Tafesse, H., Dakanay, H., Bombin, A., Wang, E., Ingersoll, J., Bifulco, K., Frediani, J. K., Parsons, R., Sullivan, J., Greenleaf, M., Waggoner, J. J., Martin, G. S., Lam, W. A., & Piantadosi, A. (2024). Respiratory virus detection and sequencing from negative SARS-CoV-2 rapid antigen tests. medRxiv : the preprint server for health sciences, 2024.08.19.24311993. https://doi.org/10.1101/2024.08.19.24311993
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: January 31, 2025
Last Modified: November 03, 2025
Protocol Integer ID: 119419
Keywords: molecular epidemiology, respiratory tract infections, public health surveillance, SARS-CoV-2, carrier RNA depletion, cDNA synthesis, Nextera, DNAse, random priming, first-strand synthesis, second-strand synthesis, metagenomic sequencing, respiratory virus detection, protocol for respiratory virus detection, nasopharyngeal swab sample, rapid antigen test, rna extraction, methods for rna, negative sar, rna, carrier rna depletion, virus, sequencing protocol
Abstract
This protocol outlines methods for RNA metagenomic sequencing to identify and characterize viruses, which we apply here to nasopharyngeal swab samples obtained from negative SARS-CoV-2 rapid antigen tests. Steps include: RNA extraction, carrier RNA depletion, cDNA synthesis (Superscript IV, ThermoFisher), and library tagmentation/indexing/amplification (Nextera XT, Illumina). Methods are based on doi: 10.3791/54117 and doi: 10.1128/mBio.01143-21 
Materials
RNA Extraction reagents


ABC
ReagentVendorCatalog
EZ1 DSP Virus KitQiagen62724



Carrier RNA depletion reagents 
ABC
Reagent  Vendor Catalog Number 
Linear Acrylamide (5 mg/ml) ThermoFisher Scientific AM9520 
NaCl (5 M), RNase-free ThermoFisher Scientific AM9760G 
Tris (1M), pH 7.0, RNase-free ThermoFisher Scientific AM9850G 
Tris (1M), pH 8.0, RNase-free ThermoFisher Scientific AM9855G 
RNAClean XP, 40 mL Beckman Coulter Life Sciences A63987 
MgCl2 (1M) ThermoFisher Scientific AM9530G 
100% EtOH Decon Labs, Inc. V1016G 
Oligo(dT)20 Primer (50 μM) (SuperScript IV First-Strand Synthesis System) ThermoFisher Scientific 18091200 
Hybridase Thermostable Rnase H Biosearch Technologies H39500 
RNase-free Buffer RDD (RNase-Free DNase Set) Qiagen 79254 
Rnase-free Dnase I (2.72 U/µl) (RNase-Free DNase Set) Qiagen 79254 
SUPERase-In Rnase Inhibitor (20 U/µl) ThermoFisher Scientific AM2694 
Nuclease-free water ThermoFisher Scientific AM9937 
ERCC RNA spike-in dilutions  NIST StoreSKU: 2374 Note: These are in-vitro transcribed RNA from ERCC plasmids sourced from NIST.

 
DNase treatment reagents 
ABC
Reagent  Vendor Catalog Number 
10X reaction Buffer (Heat&Run gDNA Removal Kit) ArcticZymes Technologies 80200-250 
HL-dsDNase (Heat&Run gDNA Removal Kit) ArcticZymes Technologies 80200-250 

 
cDNA Synthesis reagents 
ABC
Reagent  Vendor Catalog Number 
Nuclease-free waterThermoFisher ScientificAM9937
Random hexamers (50 ng/µL) (SuperScript IV First-Strand Synthesis System)ThermoFisher Scientific18091200
5X RT buffer (SuperScript IV First-Strand Synthesis System) ThermoFisher Scientific 18091200 
DTT (0.1M) (SuperScript IV First-Strand Synthesis System) ThermoFisher Scientific 18091200 
dNTP mix (10 mM) (SuperScript IV First-Strand Synthesis System) ThermoFisher Scientific 18091200 
SUPERase-In Rnase Inhibitor (20 U/µl) ThermoFisher Scientific AM2694 
SuperScript IV RT (200 U/μL) (SuperScript IV First-Strand Synthesis System) ThermoFisher Scientific 18091200 
NEBNext Second Strand Synthesis (dNTP-free) Reaction Buffer (10 X) New England Biolabs B6117S 
E. coli DNA Ligase (10 U/µl) New England Biolabs M0205L 
E. coli DNA Polymerase I (10 U/µl) New England Biolabs M0209L 
E. coli Rnase H (5 U/µl) New England Biolabs M0297L 
AMPure XP Reagent Beckman Coulter Life Sciences A63881 
Tris (1M), pH 8.0, RNase-free ThermoFisher Scientific AM9855G 
EDTA (0.5 M), pH 8.0, RNase-free ThermoFisher Scientific AM9260G 

 
Illumina Nextera XT reagents 
ABC
Reagent  Vendor Catalog Number 
Amplicon Tagment Mix (Nextera XT DNA Library Preparation Kit (96 samples)) Illumina FC-131-1096 
Tagment DNA Buffer (Nextera XT DNA Library Preparation Kit (96 samples)) Illumina FC-131-1096 
Neutralize Tagment Buffer (Nextera XT DNA Library Preparation Kit (96 samples)) Illumina FC-131-1096 
Nextera PCR Master Mix (Nextera XT DNA Library Preparation Kit (96 samples)) Illumina FC-131-1096 
IDT® for Illumina® DNA/RNA UD Indexes Sets A, B, C and D, Tagmentation (96 Indexes, 96 Samples)Illumina20027213, 20027214, 20027215, 20027216 (Note from Illumina: These index kits are "being discontinued. We will continue accepting orders until Mar 30, 2026 or until inventory is depleted." 20091654, 20091656, 20091658, and 20091660 are the recommended replacement kits, respectively.)

KAPA Library Quantification reagent kit


ABC
ReagentVendorCatalog Number
Complete kit (Universal)Roche07960140001

 
 
Troubleshooting
Before start
To minimize environmental contamination, three separate areas of the laboratory are used to: 1) prepare reagents, 2) handle samples and pre-amplified material, and 3) handle amplified material. There is a unidirectional workflow, with one individual working exclusively in the reagent area and another individual working with samples. In the protocol below, all steps are performed in the reagent area, except when noted as “sample area” or “post-amplification area".
RNA Extraction from nasopharyngeal swabs used in SARS-CoV-2 rapid antigen testing
2h

Note
This extraction protocol uses the Qiagen EZ1 Advanced XL Instrument and Qiagen EZ1 DSP Virus Kit: https://www.qiagen.com/us/products/diagnostics-and-clinical-research/solutions-for-laboratory-developed-tests/ez1-dsp-virus-kit-na


Add Linear Acrylamide to the AVE Buffer, multiplying each volume of reagent by the number of samples and overage volume of 15%:

AB
ReagentsVol. (µL)
Linear Acrylamide 3.6
AVE Buffer54
Total (µL)57.6

Extraction Protocol:

A
1. Label 2.0 mL unskirted conical-bottom sample tubes. Add 250 uL of Qiagen Buffer G2 Digestion buffer to each tube.
2. Remove the swab from the BinaxNow cartridge. Swirl it in buffer for 10 secs. Squeeze the swab on the side of the sample tube while removing the swab from the buffer.
3. Wipe the outside of the instrument with 70% EtOH, and insert the EZ1 Advanced XL DSP Virus Card into the card slot.
4. Turn on instrument and run the cleaning operation. Clean the piercing unit with 70% EtOH.
5. Press START to start work table setup. Select 200 uL sample and 60 uL elution volumes.
6. Insert loaded cartridge rack into the EZ1 instrument: a. Invert reagent cartridges (RCV) 3 times and tap the cartridges to deposit the reagents. Slide 1 cartridge rack per sample into the cartridge rack until it clicks into place.  b. Load empty 2.0 mL unskirted conical bottom tubes into the heating system slot of each cartridge.
7. Insert loaded tip rack into the EZ1 instrument: a. First row: 1.5 mL conical-bottom elution tubes b. Second row: Tip holders containing tips c. Third row: 1.5 mL conical bottom tube containing linear acrylamide and AVE buffer solution d. Fourth row: Samples from Step 2
8. Close the instrument door and press START to run EZ1 Virus Protocol.
9. Aliquot 30uL from the elution tubes into a new 1.5 mL conical bottom tube.

2h

Carrier RNA Depletion (optional step if samples were extracted with carrier RNA instead of linear acrylamide)
3h 2m
Prepare each of the following reagent mixes separately. (These volumes are sufficient for up to 40 RNA samples including any controls. If more samples are processed, increase the volume proportionately.)

Linear Acrylamide (LA) Water - make this first
AB
ReagentVol. (µl)
Linear Acrylamide 16
Nuclease-Free H2O2000

5m
1 M Tris-HCl pH 7.5 (1000 µl) - make this second
AB
ReagentVol. (µl)
Tris-HCl pH7 700
Tris-HCl pH8300

5m
5X Hybridization Buffer (100 µl)
AB
ReagentVol. (µl)
5M NaCl 20
1 M Tris-HCl (pH 7.5) 50
LA Water30

5m
10X RNase H Reaction Buffer (100 µl)
AB
ReagentVol. (µl)
1 M Tris-HCl (pH 7.5) 50
5 M NaCl 20
1 M MgCl2 20
LA Water10

5m
Prepare RNAClean Reagents:

AB
RNA SPRI Beads135 µl per sample
71% EtOH7.1 mL 100% EtOH + 2.9 mL nuclease-free water
EDTA5 µl per sample


5m
Prepare Hybridization Mix, multiplying each volume of reagent by the number of samples and overage volume of 15%:
AB
Reagents:vol. (µl)
5X Hybridization Buffer 2
LA Water0.72
Oligo dT (1500 ng stock)1
Total Volume3.72

7m
Sample Area:
A
1. Turn on thermocycler and label a 96-well microplate to hold the RNA samples.
2. Aliquot 5.28 µl of each RNA sample as well as 5.28 µl of Nuclease-free water as an NTC into the labeled microplate.
3. Add 1 µl of RNA spike-in dilutions (1 pg/µl in LA water) to each sample. (See note in Materials section regarding synthesis of these RNA controls.)
4. Add 3.72 ul of Hybridization Mix to each sample and incubate using the directions described below for Hybridization.

10m
Cycling Conditions:
AB
TempTime
95°C2 min
Ramp to 45°C-0.1°C/sec
45°Chold until next step

30m
Prepare the RNase Mix and distribute into a strip tube, multiplying each volume of reagent by the number of samples and overage volume of 15%:
AB
Reagents:vol. (µl)
10X Rnase H Reaction Buffer2
Hybridase Thermostable Rnase H (5 U/µl) 3
LA Water5
Total Volume10

5m
Sample Area:
A
1. Preheat the strip tubes containing the RNase Mix at 45°C on a heat block for 2 minutes. Warm a plate holder on another heat block at 45°C to be used for keeping the sample plate warm.
2. Remove the sample plate from the thermocycler and immediately place it in the preheated plate holder. Add 10 µl of pre-heated RNase Mix to each sample. Mix well and place back on thermocycler.
3. Incubate sample plate using the conditions described below for RNase and immediately place on ice after sample incubation concludes. Proceed directly to DNase treatment.

10m
Cycling Conditions:
AB
TempTime
45°C30 min

Prepare DNAse Mix, multiplying each volume of reagent by the number of samples and overage volume of 15%:
AB
Qiagen RDD7.5
Qiagen Rnase-free Dnase I (2.72 U/µl)2
SUPERase-In Rnase Inhibitor (20 U/µl)1
LA Water39.5
Total Volume50

5m
1. Sample Area:
A
1. Add 50 µl of DNase Mix to each sample and incubate using the conditions described below for DNase.
2. During DNase incubation, take SPRI beads out of the refrigerator to warm them to room temperature.
3. After DNase incubation finishes, add 5 µl 0.5 M EDTA to each sample to stop the reaction
4. Perform a 1.8X RNAClean XP SPRI bead clean-up

40m
Cycling Conditions:
AB
TempTime
37°C30 min

RNA SPRI Procedure:
ABCD
RNA SPRI Procedurevol. (µl)Wait time (minutes)Notes
1) Add 1.8X RNA SPRI Beads, MIX WELL1355mix halfway through
2) Place on magnet5
3) Remove buffer
4) EtOH wash2001
5) Remove EtOH
6) EtOH Wash2001
7) Remove EtOH, dry sample10wait until beads no longer appear glossy, but instead look matte (this may take fewere than 10 minutes)
8) Resuspend beads in LA Water, MIX WELL 1110mix halfway through
9) Place on magnet5
10) Remove 10 μL from each well and transfer volume to a new strip tube or 96-well plate for cDNA synthesis. Store at -80°C until use.



50m
Random Priming and cDNA Synthesis
4h 40m
Random Priming: Aliquot random primers, multiplying each volume of reagent by the number of samples and overage volume of 15%. Vortex and spin.
AB
Reagentsvol. (µl)
Random Primers  1

5m
Random Priming and cDNA Synthesis
4h 40m
Sample Area:
A
1. If samples have not undergone carrier RNA depletion, aliquot 5.28 µl of each sample into a strip tube or plate, and add 3.72 µl of water and 1 µl of RNA spike-in dilutions to each sample. If samples have undergone carrier RNA depletion, aliquot the total volume after depletion (10 µl) into a strip tube or plate, then add random primers to each sample and proceed with step 2.
2. Heat the strip tube or plate on thermocycler random priming program using the conditions described below for random priming. After incubation, immediately place samples on an ice block. Let the samples sit on the ice block for at least 1 min then spin down before next step.

15m
Cycling Conditions:
AB
TempTime
65⁰C5 min

First Strand Synthesis Mix: Make First Strand Synthesis Mix, multiplying each volume of reagent by the number of samples and overage volume of 15%.Vortex and spin:
AB
Reagentsvol. (µl)
5x SSIV Buffer4
0.1 M DTT1
10 mM dNTP mix1
dH2O1
SUPERase-In (20 U/µl)1
Superscript IV, RT (Add last)1
Total volume:9


5m
Sample Area:
A
1. Add 9 µl of First Strand Synthesis Mix to each sample.
2. Carefully mix samples with First Strand Synthesis Mix. Spin samples down and place them on thermocycler. Run the program using the conditions described below for First Strand Synthesis.
3. After sample incubation is done, spin samples for 1 minute and proceed to next step.
Cycling Conditions:

AB
Cycling Conditions
Temp. Time
23°C10 min
50°C30 min
80°C10 min

1h
Second Strand Synthesis Mix: Make Second Strand Synthesis Mix, multiplying each volume of reagent by the number of samples and overage volume of 15%. Vortex and spin:
AB
Reagents:vol. (µl)
Rnase-free water43
10X NEB Second Strand Buffer 8
10 mM dNTP mix3
E. coli DNA Ligase (10 U/µl)1
E. coli DNA Polymerase I (10 U/µl)4
E. coli Rnase H (5000 U/mL)1
Total volume:60


10m
Sample Area:
A
1. Add 60 µl of Second Strand Mix to each sample. Carefully mix and spin briefly.
2. Place samples on thermocycler and run using the conditions described below for Second Strand Synthesis.
3. Remove samples from thermocycler. Spin for 1 minute.
4. Add 5 µl EDTA to stop the reaction. Mix by pipette.
**Protocol can be paused after EDTA addition and stored at -20°C overnight** 
6. 30 minutes before 2nd strand incubation ends, remove SPRI beads from fridge to warm to room temperature. After the Second Strand incubation finishes, perform a 1.8X DNAClean XP SPRI bead clean-up.

Cycling Conditions:

AB
Temp. Time
16°C120 min

2h 15m
Prepare DNAClean Reagents:

AB
DNA SPRI Beads153 µl per sample
71% EtOH7.1 mL 100% EtOH + 2.9 mL nuclease-free water
EDTA 5 µl per sample
Elution Buffer (EB)1:100 ratio of Tris pH 8 to nuclease free water


Sample Area:

DNA SPRI Procedure:

ABCD
DNA SPRI Procedurevol. (µl)Wait time (minutes)Notes
1) Add 1.8X SPRI Beads, MIX WELL15310mix halfway through
2) Place on magnet5
3) Remove buffer
4) 71% EtOH wash1501
5) Remove EtOH
6) 71% EtOH Wash1501
7) Remove EtOH, dry sample10wait until beads no longer appear glossy, but instead look matte (this may take fewer than 10 minutes)
8) Resuspend beads in EB, MIX WELL1110mix halfway through
9) Place on magnet5
10) Aliquot 4 µl of each sample into a new strip tube or 96-well plate for Nextera. The samples can be stored at -20°C if not proceeding immediately.
11) Transfer remaining 6 µl of sample into a labelled screw-cap tube and store at -80°C.

50m
Nextera
1h 55m
Prepare Tagmentation Reagent Mix (on ice block), multiplying each volume of reagent by the number of samples and overage volume of 15%:
AB
Reagents:vol. (µl)
Amplicon Tagment Mix1
Tagment DNA Buffer5
Total volume:6
Additionally, prepare an aliquot of NT buffer at room temperature, multiplying the volume of reagent by the number of samples and overage volume of 15%:
AB
Reagents:vol. (µl)
Aliquot Neutralize Tagment Buffer (room temp)2.5
Then, prepare a mix of Nextera PCR Reagent Mix and water, multiplying each volume of reagent by the number of samples and overage volume of 15%:

AB
Reagents:vol. (µl)
NPM7.5
Water2.5
Total volume:10
Lastly, prepare DNAClean Reagents:

AB
DNA SPRI Beads42 µl per sample
71% EtOH7.1 mL 100% EtOH + 2.9 mL nuclease-free water
EB1:100 ratio of Tris pH 8 to nuclease free water


20m
Sample Area:

A
1. Turn on thermocycler.
2. Take out samples and indexes from -20°C to thaw.
3. If not previously done, aliquot 4 µl of cDNA into a 96 well plate or strip tube and place on an ice block.
4. Add 6 uL of Tagmentation Reagent Mix to each sample. Flick to mix and spin down.
5. Place on thermocycler following cycling conditions described below for Tagmentation.
6. Spin thawed tubes containing unique indexes for 2 minutes. 
7. Allow NT buffer to come to room temperature.
8. Remove samples from thermocycler and spin for 1 minute.
9. Add 2.5 µl of Neutralize Tagment (NT) buffer to stop reaction. Mix by pipette. 
10. Incubate at room temp for 5 minutes. 
11. Vortex, and centrifuge sample for 1 minute. 
12. Place samples on ice block and add 10 µl of NPM+water mix to each reaction.
13. To each sample, add 2.5 µl of a unique dual index
14. Vortex and centrifuge for 1 minute.
15. Transfer the samples on an ice block in a closed container to the post-amplification area.
16. Perform PCR with the conditions described below for the Nextera program.
17. After Nextera incubation finishes, add 35 µl of EB to each sample to bring the volume up to 60 µl, then perform a 0.7X DNAClean XP SPRI bead clean-up.


Tagmentation Cycling Conditions:

AB
TempTime
55°C5 min
10°CHold

Nextera Cycling Conditions:

ABC
TempTimeCycles
72°C3 minutes1
95°C30 sec1
95°C10 sec16 (Steps 5-7)
55°C30 sec16 (Steps 5-7)
72°C30 sec16 (Steps 5-7)
72°C5 minutes1
10°CHold
*** If low sample quality is expected, run for 18 cycles 

DNA SPRI Procedure:


ABCD
DNA SPRI Procedurevol. (µl)Wait time (minutes)Notes
1) Add 0.7X SPRI Beads, MIX WELL4210mix halfway through
2) Place on magnet 5
3) Remove buffer
4)71% EtOH wash 1501
5) Remove EtOH
6) 71% EtOH Wash 1501
7) Remove EtOH, dry sample 10wait until beads no longer appear glossy, but instead look matte
8) Resuspend beads in EB, MIX WELL 1110mix halfway through
9) Place on magnet 5
10) Transfer 10 µl eluent to a screw cap tube and store at -20°C until sequencing or pooling.

45m
Quantification, Pooling, and Sequencing
5h
KAPA Quantification:

Thaw the KAPA reaction mix and qPCR standards. Standard concentrations are as follows:

AB
KAPA Std Curve (pM)
STD 120
STD 22
STD 30.2
STD 40.02
STD 50.002
STD 60.0002
NTCNTC

Prepare the KAPA qPCR mix in a 1.5 ml tube on an ice block as listed below, multiplying the volume of each reagent by the number of samples, including the 6 standards and 1 negative control, and overage volume of 15%. Vortex and spin down.

AB
Reagentsvol. (µl)
2X Kapa Mix12
ROX LOW0.4
Total volume:12.4

Add 12.4 µl of mix to each well in an optical 96-well plate on an ice block, and then 8 µl of the NTC and 6 standards to the appropriate wells.


Sample Area: Make dilutions of the libraries with EB, and then water. Make a 1:100 dilution with EB, then make a further 1:100 dilution with water (final dilution will be 1:10,000). Add 8 µl of the final 1:10,000 dilution of each sample to the appropriate well.

Seal plate, gently vortex, and spin down. Run the plate under the following cycling conditions:
ABC
Temp. TimeCycles
95°C5 min1
95°C30 sec35
60°C45 sec35
95°C15 sec1
60°C15 sec1
95°C15 sec1


2h
Pooling: After KAPA qPCR, note the respective concentrations of each sample in nM. Normalize each sample to the desired concentration (typically 4 nM based on Illumina calculations). Make the necessary dilutions to each sample, pooling the libraries to equimolar concentrations. If the total volume of the pool is too low, the total pool volume may be brought up to 50 µl with EB. Then perform a 0.8X SPRI.

Sample Area: Perform DNA SPRI Procedure: 
 
Warm SPRI beads to room temperature (30 min) before using.

ABCD
DNA SPRI Procedurevol. (µl)Wait time (minutes)Notes
1) Add 0.8x SPRI Beads, MIX WELL4010mix halfway through
2) Place on magnet5
3) Remove buffer
4) 71% EtOH wash3001
5) Remove EtOH
6) 71% EtOH Wash3001
7) Remove EtOH, dry sample10wait until beads no longer appear glossy, but instead look matte (may take fewer than 10 min)
8) Resuspend beads in EB, MIX WELL1110mix halfway through
9) Place on magnet5
10) Transfer 10 ul of eluent to a labeled cryotube for long-term storage at -20°C.
To quantify the clean pool, perform another KAPA qPCR as outlined in Step 11 Go to , making the appropriate dilutions. Once the pool has been quantified, proceed to sequencing and prepare the pool according to manufacturer's instructions of the respective sequencing kit.


3h