TITLE: An NGS amplicon tiling protocol for HIV-1 drug resistance detection using Illumina® COVIDSeq™ Assay Kit.
AUTHORS: Noah C. Hull, Eugene Yeboah, laura.j.tsaknaridis, Vanda Makris

[DESCRIPTION]
Summary


Human immunodeficiency virus (HIV) is the pathogen responsible for acquired immunodeficiency syndrome (AIDS) and continues to be a significant global public health issue. HIV can be managed with antiretroviral (ART) drug treatments by suppressing the viral replication of HIV within the infected individual. Unfortunately, due to the nature of the virus, drug resistance can occur, making the ART drug no longer effective. An amplicon-based assay, such as the Illumina COVIDSeqTM Assay (RUO), was adopted to sequence the entire genome of the HIV-1 virus. We modified the
Illumina COVIDSeqTM Assay (RUO) protocol to prepare HIV-1 libraries and sequenced on the Illumina MiSeq. The sequencing data was analyzed by Terra.bio and Exatype NGS. This study demonstrated the utility of the assay to efficiently
sequence near-complete genome of HIV-1 virus up to 488 viral copies and provide accurate information for drug resistance detection.     

Background 

According to the World Health Organization, an estimated 39.0 million [33.1–45.7 million] people live with HIV at the end of 2022 [1]. UNAIDS reported that 40.4 million [32.9 million–51.3 million] people have died from AIDS-related illnesses since the start of the epidemic [2]. HIV drug resistance testing is crucial for managing and preventing ARV failures for persons undergoing treatment or drug-naive individuals [3]. Traditionally, HIV drug resistance testing is performed by Sanger sequencing of specific regions within the HIV genome to identify drug resistance mutations. Next-generation sequencing (NGS) technologies have recently been implemented to improve sensitivity and reproducibility and reduce the cost per sample by multiplexing. 

In this study, the Oregon State Public Health Laboratory (OSPHL), in collaboration with the Association of Public Health Laboratory (APHL), evaluated the performance of the Illumina COVIDSeqTM Assay (RUO) for HIV complete genome sequencing (HIVSeq). We describe a step-by-step HIV-1 virus genome sequencing protocol that leverages the Illumina COVIDSeqTM Assay (RUO), with reagents remaining the same.  The modified protocol utilizes HIV-1 primers designed through primal scheme. We demonstrate that whole genome sequencing of the HIV-1 virus can be achieved with extensive sequencing coverage for viral copies up to 488. Thereby expanding the use of the Illumina COVIDSeqTM Assay (RUO)
beyond SARS-CoV-2.

Results

To evaluate the performance of the assay, HIV-1 strain: IIIB positive culture control from ZeptoMetrix (Part #v0801032CF) was extracted and serially diluted to the following concentration: 10ᶺ(-1), 10ᶺ(-2), 10ᶺ(-3), 10ᶺ(-4), 10ᶺ(-5), and 10ᶺ(-6). Library preparation of the diluted viral nucleic extracts was sequenced on the Illumina MiSeq 2 x 150 bp. Viral copies ranged from 48.8 to 4,880,000 viral RNA copies/ul. We evaluated the assay's performance for accuracy, precision,
sensitivity, specificity, and limit of detection. 

HIV-1 primers
designed with Primal Scheme: Complete Primers under MATERIALS section.


To verify the coverage and detect drug-resistant mutations of all samples, we used FASTQ files exported from the MiSeq instrument. We uploaded them to the Terra platform, used the Illumina PE TheiaCov workflow developed by Theiagen, and mapped them to reference NC-001802.1 to generate run and quality metrics. FASTA files were analyzed for drug resistance mutations using Exatype NGS by Hyraxbio.  All samples were run in triplicates. Quality metrics and coverage analysis are shown in Tables 1A and 1B. The average percent reference coverage for viral loads of 4,880,000, 488,000,
48,800, 4,880, and 488 were >90%. Reference coverage for negative controls was undetected due to assembly failure. These results showed that the assay performed well in sequencing HIV-1-positive virus. 

To evaluate the accuracy of the assay, we leveraged the HIV-1 positive control’s mutations. The positive control has a non-polymorphic mutation F227L found in the non-nucleoside reverse transcription. We calculated the percent accuracy based on the ability of the assay to identify this mutation in all 17 samples that passed quality control metrics. Table 2 shows a
100% consistent detection of the F227L mutation in all samples. 

We evaluated the analytical sensitivity of the assay based on the HIV-1 positive control drug-resistant profile within the NRTI – Nucleoside Reverse Transcriptase Inhibitors (susceptible), NNRTI - Non-Nucleoside Reverse Transcriptase Inhibitors (3 intermediate and F227L mutation), PI - Protease Inhibitors (susceptible), and INSTI - Integrase Inhibitors regions (susceptible). The assay detected concordant results in 64 out of 68 drug classes and their resistance calls in all the dilution series (64/68) x 100 = 94%). Data is shown in table 4. Within-run precision was calculated based on three replicates from each dilution series (4.880,000, 488,000, 48,000, 4,800, 480, and 48.8 copies) of the HIV-1 subtype B positive control and three replicates of a negative template control. There were expected 17 positive and eight negative
 (3 negative and five non-HIV specimens). The miscalculation rate was 0%. Also, Positive Predictive Value =100% and Negative Predictive Value = 100%. Figure 1 demonstrates that sequencing the HIV-1 genome using the COVIDSeqTM  Assay (RUO) (on the Illumina MiSeq 2X150) produces >80% coverage of the complete HIV-1 genome with viral copy down to 488 copies/ul.  

Analytical specificity and coinfection for HIV-1 genome were evaluated using confirmed positive samples of HAV-Hepatitis A, HBV-Hepatitis B, HCV-Hepatitis C, and two TP-Syphilis. All samples were confirmed to be negative for HIV-1. QC metrics failed for all five samples due to genome assembly failure and no reads, as depicted in Table 6.  Another evaluation of analytical specificity of the mutation detected was calculated based on the assay’s ability to detect the
mutation, F227C, a rare nonpolymorphic mutation at position 106, at NNRTI - Non-Nucleoside Reverse Transcriptase
Inhibitors region in each of the 17 samples at the different viral loads of (4.880,000, 488,000, 48,000, 4,800,
480, and 48.8). Table 5 shows (17/17) x 100 =100.00% analytical specificity to detect the mutation.

We evaluated the Limit of Detection by analyzing all samples, their experimental/expected results, and the ability to detect
mutations and drug-resistant calls in all four drug-resistant regions of the Pol Region (NRTI, NNRTI, PI, and INSTI). Table 6 shows a 100% concordance between expected and experimental results for
viral copy number dilutions up to 10ᶺ (-5) or 488 copies. 

At 10ᶺ (-6) or 49 viral copies, sensitivity decreases to 8/12 = 67%.

These results in Table 7 indicate that positive HIV-1 samples with viral loads up to 488 copies can reliably produce a drug resistance profile. Viral copies under 488 have inconsistent results and do not pass quality control metrics. 


  *One sample from HIV_PC_1:10e4 (2) failed QC due to sample loss during library preparation.  Analysis shows inclusion and omission metrics.  Triplicate Average: Average mean calculation for dilutions (all dilutions were run in triplicate)



Table 1B. HIV-1 Positive Control Quality Control Metrics (Raw Data) Analysis on Terra.bio

  
     Dilution
  Factor      Assembly
  Length Unambiguous      Assembly
  Mean Coverage      Number
  of  Ns      Total
  Number      %
  Reference Coverage        HIV_PC_10_1      9040      6854.19      128      9175      98.46        HIV_PC_10_2      9040      5455.84      135      9182      98.46        HIV_PC_10_3      9079      11842.6      88      9174      98.89        HIV_PC_100_1      9040      6085.74      125      9172      98.46        HIV_PC_100_2      9041      6447.61      122      9169      98.48        HIV_PC_100_3      9040      4327.41      102      9149      98.46        HIV_PC_1000_1      9041      10369.2      105      9152      98.48        HIV_PC_1000_2      9040      4998.39      121      9168      98.46        HIV_PC_1000_3      9039      3504.9      105      9152      98.45        HIV_PC_10000_1      9044      14738.5      127      9174      98.51        HIV_PC_10000_2      5010      8504.21      3419      8432      54.57        HIV_PC_10000_3      8769      4044.16      375      9151      95.51        HIV_PC_100000_1      8739      5186.66      399      9142      95.19        HIV_PC_100000_2      8730      5353.17      411      9156      95.09        HIV_PC_100000_3      8129      3741.45      1014      9149      88.54        HIV_PC_1000000_1      6263      2370.05      2879      9147      68.22        HIV_PC_1000000_2      6784      2204.5      2361      9151      73.89        HIV_PC_1000000_3      6441      2068.95      2713      9156      70.16   


Table 3. HIV Drug Resistance Mutations


HIV-1 drug-resistant profiles from Exatype NGS, using the Stamford HIV drug-resistant database. 

     Dilution
  Factor      NRTI – Nucleoside Reverse Transcriptase
  Inhibitors      NNRTI - Non-Nucleoside Reverse Transcriptase
  Inhibitors      PI -
  Protease Inhibitors         INSTI -  Integrase Inhibitors        HIV_PC_10_1      Susceptible
        2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_10_2      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_10_3      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_100_1      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_100_2      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_100_3      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_1000_1      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_1000_2      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_1000_3      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_10000_1      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_10000_2      Low
  coverage      Low
  coverage      Low
  coverage      Low coverage        HIV_PC_10000_3      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_100000_1      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_100000_2      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_100000_3      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Susceptible        HIV_PC_1000000_1      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Susceptible      Low coverage        HIV_PC_1000000_2      Susceptible      2-Susceptible,
  3-Intermediate  ETR :
  [RT] F227L      Low
  coverage      Low coverage        HIV_PC_1000000_3      Susceptible      2-Susceptible,
  3-Low coverage  ETR :
  [RT] F227L      Susceptible      Low coverage   

 

Table 4: Within Run Precision


     Sensitivity      Number of
  Copies n=6/dilution      NRTI – Nucleoside Reverse Transcriptase
  Inhibitors      NNRTI - Non-Nucleoside Reverse Transcriptase
  Inhibitors      PI -
  Protease Inhibitors         INSTI -  Integrase Inhibitors        8/12      HIV_PC_10ᶺ(-6),
  _1      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Low coverage        HIV_PC_10ᶺ(-6),
  _2      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Low coverage      Low coverage        HIV_PC_10ᶺ(-6),
  _3      Susceptible      2-Susceptible, 3-Low coverage  ETR : [RT] F227L      Susceptible      Low coverage        12/12      HIV_PC_10ᶺ(-5),
  _1      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-5),
  _2      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-5),
  _3      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        *8/12  8/8      HIV_PC_10ᶺ(-4),
  _1      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-4),
  _2      Low coverage      Low coverage      Low coverage      Low coverage        HIV_PC_10ᶺ(-4),
  _3      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        12/12      HIV_PC_10ᶺ(-3),
  _1      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-3),
  _2      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-3),
  _3      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        12/12      HIV_PC_10ᶺ(-2),
  _1      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-2),
  _2      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-2),
  _3      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        12/12      HIV_PC_10ᶺ(-1),
  _1      Susceptible       2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-1),
  _2      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible        HIV_PC_10ᶺ(-1),
  _3      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Susceptible   



Table 5: Analytical Specificity for Mutation Detected:

 
     Analytical
  Specificity      Number of
  Copies n=6/dilution      NNRTI -
  Non-Nucleoside Reverse Transcriptase Inhibitors        3/3      HIV_PC_10ᶺ(-6)      ETR : [RT] F227L        3/3      HIV_PC_10ᶺ(-5)      ETR : [RT] F227L        *2/3  2/2      HIV_PC_10ᶺ(-4)      ETR : [RT] F227L        Low coverage        ETR : [RT] F227L        3/3      HIV_PC_10ᶺ(-3)      ETR : [RT] F227L        3/3      HIV_PC_10ᶺ(-2)      ETR : [RT] F227L        3/3      HIV_PC_10ᶺ(-1)      ETR : [RT] F227L   
 

Table 6: Analytical Specificity of Co-infection
 
     Accession
  #      Specimen      HIV-1
  Assay Result        7427      HAV+      No sequence        1701      HBV+      No sequence        1740      HCV+      No sequence        4023      TP+      No sequence        7214      TP+      No sequence   

Table 7: Limit of Detection Threshold


     Sensitivity      Number of
  Copies n=6/dilution      NRTI – Nucleoside Reverse Transcriptase
  Inhibitors      NNRTI - Non-Nucleoside Reverse Transcriptase
  Inhibitors      PI - Protease
  Inhibitors         INSTI -  Integrase Inhibitors        8/12      HIV_PC_10ᶺ(-6),
  _1      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Susceptible      Low coverage        HIV_PC_10ᶺ(-6),
  _2      Susceptible      2-Susceptible, 3-Intermediate  ETR : [RT] F227L      Low coverage      Low coverage        HIV_PC_10ᶺ(-6),
  _3      Susceptible      2-Susceptible, 3-Low coverage  ETR : [RT] F227L      Susceptible      Low coverage   

 

Note:

It is important to note that the positive control was a culture of a USA HIV-1 strain IIIB, and primers used for this assay were designed based on HIV-1 sequences primarily from North America. Due to the high genome variability of
the HIV-1 virus, primers must be designed using sequences in or around the region of interest. The primers used in this assay performed well, with good genome coverage on samples from North America. However, they showed poor coverage of HIV-1-positive samples from different continents. Hence, the recommendation of designing primers specific to the region to account for variations within the region to achieve higher genome coverage. 

 
References:

[1]“HIV and AIDS.” World Health Organization, World Health Organization, 13 July 2023, www.who.int/news-room/fact sheets/detail/hiv-aids. 

[2]“Global HIV & AIDS Statistics — Fact Sheet.” Www.unaids.org, 2023, www.unaids.org/en/resources/factsheet#:~:text=Global%20HIV%20statistics&text=29.8%20million%20people%20were%20accessing. Accessed 15 Oct. 2023.

[3] Günthard H.F., Calvez V., Paredes R., Pillay D., Shafer R.W., Wensing A.M., Jacobsen D.M., Richman D.D. Human
immunodeficiency virus drug resistance: 2018 recommendations of the international antiviral society-USA panel. Clin. Infect. Dis. 2019;68:177–187. doi: 10.1093/cid/ciy463

[4] “Illumina COVIDSeq RUO Kits Reference Guide (1000000126053 v08)” Illumina COVIDSeq
Research Use Only Kits Documentation. COVIDSeq Assay (96 samples) | Low-throughput COVID-19 surveillance
(illumina.com). 2022
 


[GUIDELINES]
Follow the manufacturer's specifications and guidelines.

[STEPS]
2. 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 (1.74*10^10 IU/mL or 2.09*10^10 ng/ml); Sample was diluted 1:10 = 2.09*10^9 ng/ml before extraction. cDNA Synthesis and Amplification
1. 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. 
3. 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. 
4. Prepare the following consumables: 
     Reagent      Storage      Instructions        EPH3      -25°C to -15°C      Thaw at room temperature, and then
  invert to mix   
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 
    Label new PCR plate CDNA1. 
    Add 8.5 µl EPH3 to each well. 
    Add 8.5 µl eluted sample to each well. 
    Seal and shake at 1600 rpm for 1 minute. 
    Centrifuge at 1000 × g for 1 minute. 
    Place on the preprogrammed thermal cycler and run the COVIDSeq Anneal program.
5. Synthesize First Strand cDNA:
     Reagent      Storage      Instructions        FSM      -25°C to -15°C      Thaw and bring to room temperature.
  Invert to mix, and then keep on ice.        RVT      -25°C to -15°C      Invert to mix before use. Keep on
  ice   
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

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.
6. Amplify cDNA:
Preparation:

Prepare the following consumables:
 
     Reagent      Storage      Instructions        CPP1      -25°C to -15°C      Thaw at room temperature. Keep on
  ice until use.        CPP2      -25°C to -15°C      Thaw at room temperature. Keep on
  ice until use.        IPM      -25°C to -15°C      Thaw at room temperature, and then
  invert to mix. Keep on ice until use.   
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.
 
     Reagent      COVIDSeq PCR 1 Master Mix (µl)      COVIDSeq PCR 2 Master Mix (µl)        IPM      15      15        CPP1      4.3      N/A        CPP2      N/A      4.3        Nuclease-free water      4.7      4.7   
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 POINT If you are stopping, seal the plate and store at -25°C to -15°C for up to 3 days.
7. Tagment PCR Amplicons

Preparation:

      Prepare the following consumables:
     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.
8. Post Tagmentation Clean Up:

Preparation:

1.  Prepare the following consumables:
     Reagent      Storage      Instructions        ST2      Room temperature      Vortex before use.        TWB      2°C to 8°C      Vortex before use.   

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.
9. Amplify Tagmented Amplicons:

Preparation 

      Prepare the following consumables:
     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.
10. Pool and Clean Up Libraries

Preparation:

     Prepare the following consumables:
     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.
11. 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.