1863293f4d972268d6f2967146dd110e pmc_11122360.pdf 824188d5d00189325375ba6f386ccd019bbda3c7 pmc_11122360.pdf cbd56d9861ed5535e7a4c404dd24820f14903c01aa5618d4c4e8665a258f51eb pmc_11122360.pdf Title: Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation Subject: Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5' end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A–H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies. Keywords: hepatitis B virus; genotypes; next-generation sequencing; quasispecies; conservation; information content; Distance-Based discrimination method; preS; hepatitis B X gene; RNA interference Author: Eva Dopico, Marta Vila, David Tabernero, Josep Gregori, Ariadna Rando-Segura, Beatriz Pacín-Ruíz, Laura Guerrero, Itziar Ubillos, Miguel J. Martínez, Josep Costa, Josep Quer, Javier Pérez-Garreta, Alejandra González-Sánchez, Andrés Antón, Tomás Pumarola, Mar Riveiro-Barciela, Roser Ferrer-Costa, Maria Buti, Francisco Rodríguez-Frías and Maria Francesca Cortese Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri May 17 12:37:32 2024 CEST ModDate: Fri May 17 12:41:47 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 23 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4093751 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JYPLJJ+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 IFKYFA+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 QZWRAX+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 BHTRKU+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 NVRAIR+CMSY10 Type 1 Builtin yes yes yes 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Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Hepatitis B Virus Genomic Regions Analyzed and Genotype Determination Using the Distance-Based Discrimination Method Destination: subsection.2.1 Item: Title: Analysis of Hepatitis B Virus Genotypes Destination: subsection.2.2 Children: Item: Title: Next-Generation Sequencing Yield and Haplotype Genotyping Destination: subsubsection.2.2.1 Item: Title: Analysis of Main Hepatitis B Virus Genotypes and Genotype Mixtures in Chronic Hepatitis B Patients Destination: subsubsection.2.2.2 Item: Title: Study of Sequence Conservation in the preS and 5X Amplicons across Different Genotypes Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Patients and Samples Destination: subsection.4.1 Item: Title: Serological and Virological Determinations Destination: subsection.4.2 Item: Title: Amplification and Next-Generation Sequencing of the Hepatitis B Virus Genome Destination: subsection.4.3 Item: Title: Sequencing Hepatitis B Virus Amplicons with the Sanger Method Destination: subsection.4.4 Item: Title: Bionformatics Analysis of Sequencing Data Destination: subsection.4.5 Children: Item: Title: Processing the Next-Generation Sequencing Raw Data Destination: subsubsection.4.5.1 Item: Title: HBV Genotype Determination with the Distance-Based Discrimination Method and Selection of Reference Sequences Destination: subsubsection.4.5.2 Item: Title: Quasispecies Sequence Conservation Destination: subsubsection.4.5.3 Item: Title: Statistical Methods Destination: subsection.4.6 Item: Title: References Destination: appendix.A. Info: Title: Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation Author: Eva Dopico, Marta Vila, David Tabernero, Josep Gregori, Ariadna Rando-Segura, Beatriz Pacín-Ruíz, Laura Guerrero, Itziar Ubillos, Miguel J. Martínez, Josep Costa, Josep Quer, Javier Pérez-Garreta, Alejandra González-Sánchez, Andrés Antón, Tomás Pumarola, Mar Riveiro-Barciela, Roser Ferrer-Costa, Maria Buti, Francisco Rodríguez-Frías and Maria Francesca Cortese Subject: Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5' end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies. 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