NGS amplification panel for HBV (Hepadnaviridae: Orthohepadnavirus) sequencing

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Abstract

Introduction. Hepatitis B virus (HBV) remains a pressing global public health concern. The clinical course of the disease, particularly its tendency towards chronicity and response to therapy, is significantly influenced by the HBV genotype and specific mutations. There is an imperative need for a straightforward, highly sensitive, and dependable method for whole genome sequencing of HBV.

Objective. Development and testing of an amplification panel for HBV whole-genome sequencing.

Materials and methods. We introduce an NGS amplification panel designed for genome sequencing of HBV on the Illumina platform. A panel consisting of 54 primers, divided into 2 pools and amplifying overlapping regions of the HBV genome up to 300 bp in length, was tested on 246 HBV DNA samples.

Results. The studied samples represented a genotypic diversity of the virus, with a pronounced predominance of the genotype specific to the Moscow region: 216, 27, 2, and 1 sample were identified as genotype D, A, B, and E, respectively. Five samples contained at least one mutation associated with antiviral therapy resistance, and twenty-three samples contained at least one mutation associated with vaccine escape described in the literature.

Conclusion. The present paper describes the stages of whole-genome sequencing of HBV, provides a laboratory protocol, nucleotide sequences of the primers and an approach to the data analysis. Using a list of clinical samples as example, the reliability of the panel is shown. The HBV panel holds immense potential for utilization in scientific research, epidemiological monitoring, and advancement of personalized medicine approaches.

About the authors

Mikhail D. Chanyshev

Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Author for correspondence.
Email: chanishq@gmail.com
ORCID iD: 0000-0002-6943-2915

PhD (Biol.), Researcher of the Laboratory for genomic research, Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Russian Federation, 111123, Moscow

Natalia V. Vlasenko

Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Email: vlasenko@cmd.su
ORCID iD: 0000-0002-2388-1483

Researcher of the Laboratory of Viral Hepatitis, Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Russian Federation, 111123, Moscow

German V. Roev

Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing; Moscow Institute of Physics and Technology, National Research University

Email: roevherman@gmail.com
ORCID iD: 0000-0002-2353-5222

биоинформатик Лаборатории геномных исследований ФБУН ЦНИИ Эпидемиологии Роспотребнадзора

Russian Federation, 111123, Moscow; 115184, Dolgoprudny

Ivan A. Kotov

Moscow Institute of Physics and Technology, National Research University

Email: ivan.kotov@phystech.edu
ORCID iD: 0000-0003-2416-5689

PhD student, Phystech School of biological and medical physics of MIPT

Russian Federation, 115184, Dolgoprudny

Albina G. Glushchenko

Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing; Moscow Institute of Physics and Technology, National Research University

Email: albinagluschenko@gmail.com
ORCID iD: 0009-0002-8851-8703

laboratory assistant of the Laboratory for genomic research, Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Russian Federation, 111123, Moscow; 115184, Dolgoprudny

Vera V. Makashova

Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Email: veramakashova@yandex.ru
ORCID iD: 0000-0002-0982-3527

Doctor of Medical Sciences, Professor, Leading Researcher of Clinical Department of Infectious Pathology Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Russian Federation, 111123, Moscow

Kamil F. Khafizov

Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Email: kkhafizov@gmail.com
ORCID iD: 0000-0001-5524-0296

PhD (Biol.), Head of the Laboratory for genomic research, Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Russian Federation, 111123, Moscow

Vasily G. Akimkin

Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Email: vgakimkin@yandex.ru
ORCID iD: 0000-0003-4228-9044

Academician of the Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Director of Central Research Institute for Epidemiology of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing

Russian Federation, 111123, Moscow

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Supplementary files

Supplementary Files
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1. JATS XML
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2. Fig. 1. Log10 transformed read counts per amplicon norma by total reads per sample. HBV genome was divided into 20 amplicons (HBV-1–HBV-20).

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3. Fig. 2. Coverage of 246 samples. The genome region was considered as covered at a minimum depth of 10 reads. Samples are sorted by coverage. The color designation of HBV genotypes corresponds to that from S. Velkov et al. [6].

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4. Fig. 3. Occurrence of various HBV genotypes in the Moscow region of Russia. The color designation of HBV genotypes corresponds to that from [6].

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5. Fig. 4. Phylogenetic tree for sequenced HBV samples. The tree was constructed in MEGA v11.0.11 using the neighbor joining algorithm and rooted using the midpoint method. Visualization was made using the ete3 library. Genotypes A, B, D, E are marked in red, blue, violet, and orange, respectively. References and sequences from this study are highlighted in black and yellow, respectively.

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6. Fig. 5. The detection rates of mutations in RT domain of P gene associated with resistance to antiviral therapy and mutations in S gene associated with evasion of vaccine-induced response. Gray, white, yellow, and red boxes represent respectively the undetermined regions, wild type, substitutions not described in the literature, and substitutions described in the literature data as associated with resistance to antiviral therapy and vaccine escape mutations. Total 33 samples containing at least one difference from the wild type are presented.

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7. Appendix
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Copyright (c) 2024 Chanyshev M.D., Vlasenko N.V., Roev G.V., Kotov I.A., Glushchenko A.G., Makashova V.V., Khafizov K.F., Akimkin V.G.

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This work is licensed under a Creative Commons Attribution 4.0 International License.

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