Журнал микробиологии, эпидемиологии и иммунобиологии. 2021; 98: 627-638
Применение секвенирования следующего поколения для исследования двойной ВИЧ-инфекции
https://doi.org/10.36233/0372-9311-153Аннотация
Введение. Целью исследования был сравнительный анализ эффективности выявления и подтверждения двойной ВИЧ-инфекции с применением классического популяционного секвенирования (ПС) и секвенирования следующего поколения (NGS) для фрагмента гена pol ВИЧ-1, кодирующего протеазу и часть обратной транскриптазы (позиции 2253–3368).
Материалы и методы. Исследованы модельные образцы межсубтиповой двойной ВИЧ-инфекции, содержащие вирусы ВИЧ-1 субтипа B, субсубтипа A6 и рекомбинантной формы CRF63_02A1. Вирусы смешивали попарно в соотношении от 10 до 90% для создания 3 групп модельных образцов: CRF63vsB, CRF63vsA6 и A6vsB. Нуклеотидные последовательности, полученные технологиями ПС и NGS, с порогами чувствительности к минорным вариантам вируса 5, 10, 15 и 20% (NGS5–NGS20 соответственно) использовали для определения величин индекса вырожденности (DB) и индекса синонимичности (SM). Фрагмент исследуемого региона (позиции 2725–2981) служил для анализа операционных таксономических единиц.
Результаты. Применение NGS5 оказалось наиболее эффективным для выявления двойной ВИЧ-инфекции в модельных образцах. Было обнаружено статистически достоверное (p < 0,01) увеличение DB- и SM-индексов для NGS5 по сравнению с ПС. Это позволило с помощью NGS5 выявить двойную ВИЧ-инфекцию в 25 модельных образцах из 27, в то время как ПС позволяло выявлять её лишь в 15 образцах. Анализ операционных таксономических единиц подтвердил двойную ВИЧ-инфекцию во всех группах модельных образцов.
Обсуждение. Эффективность выявления и подтверждения двойной ВИЧ-инфекции зависит как от доли каждого вируса в образце, так и от генетических особенностей данных вирусов. Заключение. Внедрение в рутинную практику генетического анализа технологии NGS позволит не только более эффективно выявлять генетические особенности инфекционных агентов, но и проводить более глубокий анализ эпидемиологической ситуации.
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Journal of microbiology, epidemiology and immunobiology. 2021; 98: 627-638
Application of next generation sequencing in dual HIV infection studies
https://doi.org/10.36233/0372-9311-153Abstract
Introduction. The aim of the study was to use comparative analysis for assessing efficiency of detection and confirmation of dual HIV infection, using conventional population sequencing (PS) and next generation sequencing (NGS) for an HIV-1 pol gene fragment, which encompasses protease and partially reverse transcriptase (positions 2253–3368).
Materials and methods. The study was performed on intersubtype dual HIV infection model samples containing viruses of HIV-1 subtype B, sub-subtype A6 and recombinant form CRF63_02A1. Viruses were mixed pairwise in proportions from 10 to 90% to obtain 3 groups of model samples: CRF63vsB, CRF63vsA6, and A6vsB. The nucleotide sequences obtained by using PS and NGS technologies having 5, 10, 15, and 20% sensitivity thresholds for minor virus variants (NGS5–NGS20, respectively) were used to estimate the number of degenerate nucleotides or the degenerate base (DB) count and the number of synonymous mutations (SM) or the SM count. The fragment of the studied region (positions 2725–2981) was used for the analysis of operational taxonomic units.
Results. The application of NGS5 proved highly efficient for detection of dual HIV infection in the model samples. The statistically significant (p < 0.01) increase in DB and SM counts was demonstrated by NGS5 compared to PS. As a result, NGS5 helped detect dual HIV infection in 25 out of 27 model samples, while with PS it was detected only in 15 samples. The analysis of operational taxonomic units confirmed dual HIV infection in all the groups of model samples.
Discussion. The efficiency of detection and confirmation of dual HIV infection depends both on the content of each virus in the sample and on genetic characteristics of these viruses. Conclusion. Using NGS genetic testing in routine practice will be instrumental for efficient identification of genetic characteristics of infectious agents and for thorough analysis of the epidemiological situation.
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