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Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Genome Amplicons.
Nolan, David J; DaRoza, Jonathan; Brody, Robin; Ganta, Krishna; Luzuriaga, Katherine; Huston, Chris; Rosenthal, Simon; Lamers, Susanna L; Rose, Rebecca.
Afiliación
  • Nolan DJ; Bioinfoexperts LLC, Thibodaux, Louisiana, USA.
  • DaRoza J; Bioinfoexperts LLC, Thibodaux, Louisiana, USA.
  • Brody R; Molecular Medicine, UMass Chan Medical School, Worcester, Massachusetts, USA.
  • Ganta K; Molecular Medicine, UMass Chan Medical School, Worcester, Massachusetts, USA.
  • Luzuriaga K; Molecular Medicine, UMass Chan Medical School, Worcester, Massachusetts, USA.
  • Huston C; Bioinfoexperts LLC, Thibodaux, Louisiana, USA.
  • Rosenthal S; Bioinfoexperts LLC, Thibodaux, Louisiana, USA.
  • Lamers SL; Bioinfoexperts LLC, Thibodaux, Louisiana, USA.
  • Rose R; Bioinfoexperts LLC, Thibodaux, Louisiana, USA.
Article en En | MEDLINE | ID: mdl-38940749
ABSTRACT
Our goal was to assess the accuracy of next generation sequencing (NGS) compared with Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n = 30) were sequenced with Sanger or reamplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB). For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical (n = 9) or nearly identical (n = 14) compared with Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had ≥35 ambiguous calls in five amplicons and 0 ambiguities in two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template) and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2-13 differences compared with Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared with Sanger and, importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates.
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Texto completo: 1 Banco de datos: MEDLINE Idioma: En Revista: AIDS Res Hum Retroviruses Asunto de la revista: SINDROME DA IMUNODEFICIENCIA ADQUIRIDA (AIDS) Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos

Texto completo: 1 Banco de datos: MEDLINE Idioma: En Revista: AIDS Res Hum Retroviruses Asunto de la revista: SINDROME DA IMUNODEFICIENCIA ADQUIRIDA (AIDS) Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos