{
  "abstract": "Background Whole-genome sequencing (WGS) of HIV-1 permits the comprehensive characterization of viral diversity, drug-resistance mutations, and transmission dynamics beyond the limits of pol sequencing. However, its implementation in clinical practice remains challenging.Material and Methods HIV-1 positive plasma samples were processed for WGS using the QIAseq xHYB HIV-1 kit (Qiagen) and sequenced on the MiSeq platform (Illumina). A fully reproducible analysis pipeline was implemented in Snakemake. Preliminary consensus genomes were generated using the HIV-1 reference K03455 and queried by BLAST against a custom database of complete HIV-1 genomes to select the best sample-specific reference. Consensus sequences and SNP tables were rebuilt using this reference. Variant calling was performed with LoFreq using a 1% allele frequency cut-off. Consensus sequences were aligned with 555 HIV-1 reference genomes, and subtype assignment was performed by maximum-likelihood phylogenetic inference in IQ-TREE2 under the GTR+F+R9 model. Drug resistance was also assessed.Results Nineteen HIV-1 RNA samples from drug-naïve individuals living with HIV with a median viral load of 3.05x10 5 copies/mL (interquartile range [IQR]: 5.51x104-9.98x105 copies/mL) were processed and analyzed by WGS. Sequencing performance demonstrated high overall genome recovery across samples, but for two (a B and a CRF02 subtype). The median genome coverage was 5,926x (IQR: 920x-45,961x). Median genome coverage at >100× depth was 95.15% (IQR: 88.13%–97.20%). A median of 3.87% of bases was at zero coverage (IQR: 1.06%-7.33%). Low-coverage regions were consistently observed at nucleotide positions 7110–7468 (K03455 coordinates), indicating systematic limitations in the env V3–V5 region. Phylogenetic analysis showed subtype B in 5 samples (29.4%), followed by CRF02_AG (4, 23.5%), CRF01_AE (2, 11.8%), and single representatives of A1, A6, CRF66_BF1, CRF46_BF1, 19_CPX, and 121_0107CN. Phylogeny confirmed pol-based subtype assignment for all samples except for an F1 pol sequence becoming a CRF66_BF1. Eight (47.1%) samples carried at least one resistance-associated mutation. While no major PI or INSTI resistance mutations were found, NRTI- and NNRTI-associated mutations were observed in 2 (11.8%) and 4 (23.5%) samples each. Outside pol, WGS identified two dolutegravir-associated mutations in one 66_BF1 and one B sample (env A539V and Y61H). Fostemsavir-associated gp120 substitutions were detected in 5 samples (env M426L in one CRF66_BF1, M434I in one A1 and one CRF02_AG, M475I in one CRF01_AE, and M426R in one B), while only the accessory mutation to Lenacapavir T107A in gag was found in two CRF02_AG samples.Conclusions WGS enables comprehensive HIV-1 characterization beyond pol. Low coverage in env regions indicates the need for improved enrichment and analytical strategies to ensure complete genome reconstruction for clinical and surveillance applications.",
  "authors": [
    {
      "affiliations": [
        "Microbiology and Virology Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "A Callegaro"
    },
    {
      "affiliations": [
        "Microbiology and Virology Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "M Mazzoni"
    },
    {
      "affiliations": [
        "Microbiology and Virology Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "S Uceda Renteria"
    },
    {
      "affiliations": [
        "Infectious Diseases Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "G Bozzi"
    },
    {
      "affiliations": [
        "Microbiology and Virology Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "A Parisi"
    },
    {
      "affiliations": [
        "Infectious Diseases Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "V Castelli"
    },
    {
      "affiliations": [
        "Infectious Diseases Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "B Mariani"
    },
    {
      "affiliations": [
        "Infectious Diseases Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy"
      ],
      "name": "A Muscatello"
    },
    {
      "affiliations": [
        "Infectious Diseases Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy",
        "Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy"
      ],
      "name": "A Bandera"
    },
    {
      "affiliations": [
        "Microbiology and Virology Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy",
        "Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy"
      ],
      "name": "C Alteri"
    }
  ],
  "title": "P5 Performance of HIV-1 whole-genome sequencing in HIV-RNA positive plasma samples",
  "uid": "6868e4a4-5a12-5572-94d7-5639ecd9f9b8"
}