An NGS amplicon tiling protocol for HIV-1 drug resistance detection using Illumina® COVIDSeq™ Assay Kit.

Sequencing libraries were prepared using reagents in the Illumina COVIDSeq Assay (RUO) kit with a protocol modified by substituting SARS-CoV-2 primers with HIV-1 primers.

Sample Extraction

HIV-1 subtype B positive culture

control ZeptoMetrix (Part #v0801032CF), was extracted via the Qiagen Advanced XL DSP kit on the EZ1 extractor and serially diluted to the following concentration: 10ᶺ(-1), 10ᶺ(-2), 10ᶺ(-3), 10ᶺ(-4), 10ᶺ(-5), and 10ᶺ(-6), Undiluted control (1.7410^10 IU/mL or 2.0910^10 ng/ml); Sample was diluted 1:10 = 2.09*10^9 ng/ml before extraction. cDNA Synthesis and Amplification n

Prepare the HIV-1 primer pool as described by the manufacturer. Individual primers were obtained from IDT as desalted, lab-ready stock at 100mM concentration. Dilute each primer at ratios provided by the manufacturer. Dilute the primer pool to get a 10 µM working concentration.

Prepare the following consumables:

Save the following COVIDSeq Anneal program on the thermal cycler:

• Choose the preheat lid option
• Set the reaction volume to 17 µl
• 65°C for 3 minutes
• Hold at 4°C

Procedure

1. Label new PCR plate CDNA1.
2. Add 8.5 µl EPH3 to each well.
3. Add 8.5 µl eluted sample to each well.
4. Seal and shake at 1600 rpm for 1 minute.
5. Centrifuge at 1000 × g for 1 minute.
6. Place on the preprogrammed thermal cycler and run the COVIDSeq Anneal program.

Synthesize First Strand cDNA:

Save the following COVIDSeq FSS program on the thermal cycler:

• Choose the preheat lid optio.n
• Set the reaction volume to 25 µl
• 25°C for 5 minutes
• 50°C for 10 minutes
• 80°C for 5 minutes
• Hold at 4°C

Procedure

1. In a 1.7 ml tube, combine the following volumes to prepare First Strand cDNA Master Mix. Multiply each volume by the number of samples.

• FSM (9 µl)
• RVT (1 µl) Reagent overage is included to account for small pipetting errors.

2. Add 8 µl master mix to each well of the CDNA1 plate.

3. Seal and shake at 1600 rpm for 1 minute.

4. Centrifuge at 1000 × g for 1 minute.

5. Place on the preprogrammed thermal cycler and run the COVIDSeq FSS program

SAFE STOPPING POINT:   If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

Amplify cDNA:

Preparation:

1. Prepare the following consumables:

2. Save the following COVIDSeq PCR program on the thermal cycler:

• Choose the preheat lid option

• Set the reaction volume to 25 µl

• 98°C for 3 minutes

• 35 cycles of:   98°C for 15 seconds 63°C for 5 minutes

• Hold at 4°C

Procedure:

1. Label two new PCR plates COV1 and COV2. The plates represent two separate PCR reactions on

each sample and control in the CDNA1 plate.

2. In a 15 ml tube, combine the following volumes to prepare COVIDSeq PCR 1 Master Mix and

COVIDSeq PCR 2 Master Mix. Multiply each volume by the number of samples. Reagent overage is included to account for small pipetting errors.

3. Add 20 µl COVIDSeq PCR 1 Master Mix to each well of the COV1 plate corresponding to each well of the CDNA1 plate.

4. Add 5 µl first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of the COV1 plate.

5. Add 20 µl COVIDSeq PCR 2 Master Mix to each well of the COV2 plate corresponding to each well of the CDNA1 plate.

6. Add 5 µl first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of the COV2 plate.

7. Seal and shake at 1600 rpm for 1 minute.

8. Centrifuge at 1000 x g for 1 minute.

9. Place in the preprogrammed thermal cycler and run the COVIDSeq PCR program.

SAFE STOPPING POIN T If you are stopping, seal the plate and store at -25°C to -15°C for up to 3 days.

Tagment PCR Amplicons

Preparation:

1. Prepare the following consumables: | A | B | C | | --- | --- | --- | | Reagent | Storage | Instructions | | EBLTS | 2°C to 8°C | Bring to room temperature. Vortex thoroughly before use. | | TB1 | -25°C to -15°C | Bring to room temperature. Vortex thoroughly before use. |

2. If COV1 and COV2 plates were stored frozen, prepare as follows.

a.  Thaw at room temperature.

b. Check seals, and then shake at 1600 rpm for 1 minute.

c. Centrifuge at 1000 x g for 1 minute.

3. Save the following COVIDSeq TAG program on the thermal cycler:

• Choose the preheat lid option
• Set the reaction volume to 50 µl
• 55°C for 5 minutes
• Hold at 10°C

Procedure:

1. Label a new PCR plate TAG1.

2. Combine COV1 and COV2 as follows.

   a. Transfer 10 µl from each well of the COV1 plate to the corresponding well of the TAG1 plate.

   b. Transfer 10 µl from each well of the COV2 plate to each well of the TAG1 plate containing COV1.

3. In a 15 ml tube, combine the following volumes to prepare Tagmentation Master Mix. Multiply each volume by the number of samples.

• TB1 (12 µl)
• EBLTS (4 µl)
• Nuclease-free water (20 µl)

4. Add 30 µl master mix to each well in TAG1 plate.

5. Seal and shake at 1600 rpm for 1 minute.

6. Place on the preprogrammed thermal cycler and run the COVIDSeq TAG program.

Post Tagmentation Clean Up:

Preparation:

1. Prepare the following consumables:

Procedure:

1. Centrifuge the TAG1 plate at 500 x g for 1 minute.

2. Add 10 µl ST2 to each well of the TAG1 plate.

3. Seal and shake at 1600 rpm for 1 minute.

4. Incubate at room temperature for 5 minutes.

5. Centrifuge at 500 × g for 1 minute.

6. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).

7. Inspect for bubbles on the seal. If present, centrifuge at 500 x g for 1 minute, and then place on the magnetic stand (~3 minutes).

8. Remove and discard all supernatant

9. Wash beads as follows.

   a. Remove from the magnetic stand.

   b. Add 100 µl TWB to each well.

   c. Seal and shake at 1600 rpm for 1 minute.

   d. Centrifuge 500 × g for 1 minute.

   e. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).

   f. For first wash only, remove and discard all supernatant from each well.

10. Wash beads a second time. Leave supernatant in plate for second wash to prevent beads from overdrying.

Amplify Tagmented Amplicons:

Preparation

1. Prepare the following consumables: | A | B | C | | --- | --- | --- | | Reagent | Storage | Instructions | | EPM | -25°C to -15°C | Invert to mix. Keep on ice until use | | Index adapters | -25°C to -15°C | Thaw at room temperature. Vortex to mix, and then centrifuge at 1000 × g for 1 minute. |

2. Open each prepared index adapter plate seal as follows. Use a new

PCR plate for each different index set.

a. Align a new 96-well PCR plate above the index adapter plate, and then press down to puncture the foil seal.

b. Discard the PCR plate.

3. Save the following COVIDSeq TAG PCR program on the thermal

cycler:

• Choose the preheat lid option and set to 100°C

• Set the reaction volume to 50 µl

• 72°C for 3 minutes

• 98°C for 3 minutes

• 7 cycles of: o 98°C for 20 seconds

            o  60°C for 30 seconds
  
            o  72°C for 1 minute
  
            o  72°C for 3 minutes 
  

• Hold at 10°C

Procedure:

1. In a 15 ml tube, combine the following volumes to prepare PCR

Master Mix. Multiply each volume by the number of samples.

• EPM (24 µl)
• Nuclease-free water (24 µl)

2. Vortex PCR Master Mix to mix.

3. Keep the TAG1 plate on magnetic stand and remove TWB.

4. Use a 20 µl pipette to remove any remaining TWB.

5. Remove the TAG1 plate from the magnetic stand.

6. Add 40 µl PCR Master Mix to each well.

7. Add 10 µl index adapters to each well of the PCR plate.

8. Seal and shake at 1600 rpm for 1 minute.

9. If liquid is visible on the seal, centrifuge at 500 x g for 1 minute.

10. Inspect to make sure beads are resuspended. To resuspend, set your pipette to 35 µl with the plunger down, and then slowly pipette to mix.

11. Place on the preprogrammed thermal cycler and run the COVIDSeq TAG PCR program.

Pool and Clean Up Libraries

Preparation:

1. Prepare the following consumables: | A | B | C | | --- | --- | --- | | Reagent | Storage | Instructions | | ITB or IPB | Room temperature | Vortex thoroughly to mix. | | RSB | 2°C to 8°C | Let stand for 30 minutes to bring to room temperature. Vortex and invert to mix |

2. Prepare 2.5 ml 80% EtOH from absolute EtOH for each tube of pooled libraries.

Procedure for Illumina COVIDSeq Assay (96 Samples):

1. Centrifuge the TAG1 plate at 500 × g for 1 minute.

2. Place on the magnetic stand and wait until the

liquid is clear (~3 minutes).

3. To pool libraries, complete the following steps

appropriate for your kit. Repeat the steps for each additional sample plate.

a. Label a new 1.7 ml tube Pooled IPB. b

b. Transfer 5 µl library from each well of the TAG 1 plate into the Pooled IPB tube.

4. Vortex the Pooled IPB tubes to mix, and then centrifuge briefly.

5. Vortex IPB to resuspend.

6. Add IPB using the resulting volume of Pooled IPB tube volume multiplied by 0.9. For example, for 96 samples, add 432 µl IPB to each tube.

7. Vortex to mix.

8. Incubate at room temperature for 5 minutes.

9. Centrifuge briefly.

10. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).

11. Remove and discard all supernatant.

12. Wash beads as follows.

a. Keep on the magnetic stand and add 1000 µl fresh 80% EtOH to each tube.

b. Wait 30 seconds.

c. Remove and discard all supernatant.

13. Wash beads a second time.

14. Use a 20 µl pipette to remove all residual EtOH.

15. Add 55 µl RSB.

16. Vortex to mix, and then centrifuge briefly.

17. Incubate at room temperature for 2 minutes.

18. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

19. Transfer 50 µl supernatant from each Pooled IPB tube to a new microcentrifuge tube.

SAFE STOPPING POINT If you are stopping, cap the tube and store at -25°C to -15°C for up to 30 days.

Quantify and Normalize Libraries

1. Analyze 2 µl library pool using a Qubit dsDNA HS Assay kit. If libraries are outside the standard range, dilute to 1:10 concentration and analyze again.

2. Dilute each library pool to a minimum of 30 µl at a normalized concentration 4 nM using RSB according to manufacture specification.

Pool and Dilute Libraries:

After diluting to the starting concentration of 4 nM, libraries are ready to be denatured and diluted

to the final loading concentration according to the specifications of the Illumina sequencing platform.