Concurrent Nanopore Next-Generation Sequencing of Hepatitis B and Delta Virus Genomes Directly From Patient Plasma

Colson, Philippe; Boschi, Céline; Bengone-Abogourin, Jessica Grace; Bréchard, Ludivine; Motte, Anne; Allemand, Isabelle

doi:10.3343/alm.2021.41.6.608

Cited by 2 publications

(1 citation statement)

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“…HBV full genome sequencing studies, or those exploring the HBV transcriptome, have largely employed PCR- or amplicon-based deep sequencing methods ( Betz-Stablein et al, 2016 ; Sauvage et al, 2018 ; McNaughton et al, 2019 ; Astbury et al, 2020 ; Lim et al, 2021 ; Matsuda et al, 2021 ; Maslac et al, 2022 ). To date, PacBio long-read sequencing has been used to analyze the HBV transcriptome within cultured cells ( Ng et al, 2023 ), but there is only one account of ONT sequencing of HBV nucleic acid extracted directly from patient plasma without the use of PCR amplification ( Colson et al, 2021 ); however, very few HBV reads were obtained. While PCR-free RNA sequencing methods present the least bias ( Chen et al, 2021 ; De Paoli-Iseppi et al, 2021 ), they have been shown to be unsuitable for samples with low target RNA quantities and high rates of fragmentation ( Vacca et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing

Seo

Patel

et al. 2023

Front. Microbiol.

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IntroductionSerum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype (gt, A to F) and HBeAg status.MethodsLongitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients (73.3% female, 84.6% Asian) was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spike-in RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow.ResultsAll chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3′ truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3′ truncated pgRNA variants over time.ConclusionsThis study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response.

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Section: Discussionmentioning

confidence: 99%

Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing

Seo

Patel

et al. 2023

Front. Microbiol.

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Evaluation of a Next Generation Sequencing Assay for Hepatitis B Antiviral Drug Resistance on the Oxford Nanopore System

Payne,

Ritchie,

Lawson

et al. 2024

Preprint

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Background: Next-generation sequencing (NGS) for Hepatitis B virus (HBV) antiviral resistance (AVR) testing is a highly sensitive diagnostic method, able to detect low-level mutant subpopulations. Our clinical virology laboratory previously transitioned from DNA hybridization (INNO-LiPA) to NGS, initially with the GS Junior System and subsequently the MiSeq. The Oxford Nanopore Technology (ONT) sequencing system was evaluated for HBV resistance testing, with regards to sequencing accuracy and turn-around time. Methods: We performed amplicon sequencing of the HBV polymerase gene from patient plasma and external quality assessment (EQA) samples on the MiSeq Reagent Nano Kit v2 and GridION ONT with R10.4.1 flowcells. Mutational analysis and genotyping were performed by DeepChekAssay-HBV (version 2.0). Results: A total of 49 patient samples and 15 EQA samples were tested on both the MiSeq and ONT. There was high agreement for both patient and EQA samples between the MiSeq and ONT systems, with regards to total drug resistance mutations detected and total patient sample agreement, 68/70 (97%) and 47/49 (96%), respectively. Conclusion: The ONT NGS platform provided accurate HBV AVR results, with improved turn-around times. Sequencing error rates at AVR codons were below 1%.

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Concurrent Nanopore Next-Generation Sequencing of Hepatitis B and Delta Virus Genomes Directly From Patient Plasma

Cited by 2 publications

References 9 publications

Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing

Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing

Evaluation of a Next Generation Sequencing Assay for Hepatitis B Antiviral Drug Resistance on the Oxford Nanopore System

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