An NGS amplicon tiling protocol for HIV-1 drug resistance detection using Illumina® COVIDSeq™ Assay Kit. v1
Authors: C. Hull Noah 
Published: December 2023

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 theIllumina 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 efficientlysequence 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 primersdesigned 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 a100% 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 themutation, F227C, a rare nonpolymorphic mutation at position 106, at NNRTI - Non-Nucleoside Reverse TranscriptaseInhibitors 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 detectmutations 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 forviral 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 ofthe 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. Humanimmunodeficiency 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 COVIDSeqResearch Use Only Kits Documentation. COVIDSeq Assay (96 samples) | Low-throughput COVID-19 surveillance(illumina.com). 2022