Complete Hepatitis B Virus Sequencing using an ONT-Based Next-Generation Sequencing Protocol v1

Résumé

This protocol outlines the process of generating complete Hepatitis B virus (HBV) primers from DNA extracts for next-generation sequencing using Oxford Nanopore Technology (ONT). Specifically, we have designed pan-genotypic tiling primers to cover the entire HBV genome for sequencing. The hands-on time required for a batch of 48 samples is minimal, approximately 1 hour and 30 minutes. This protocol is straightforward and can be easily adapted in settings where the ONT protocol for SARS-CoV-2 has been implemented. We provide detailed instructions for PCR amplification using HBV-specific tiling primers, sample pooling, library construction using the Rapid Barcoding Kit (SQK-RBK110.96), quantification using Qubit, and subsequent sequencing on the GridION platform. The Rapid barcoding protocol for up-to 96 samples "PCR tiling of SARS-CoV-2 virus with rapid barcoding (SQK-RBK110.96)" https://community.nanoporetech.com/protocols/pcr-tiling-of-sars-cov-2-virus-with-rapid-barcoding-sqk-rbk110/v/PCTR_9125_v110_revA_24Mar2021. This protocol describes a modified version of the 1200bp amplicon "midnight" primer set for nCoV-2019 (SARS-CoV-2) amplification, utilizing the Nanopore Rapid kit for MinION. The modification was developed by Nikki Freed. https://dx.doi.org/10.17504/protocols.io.bwyppfvn and the original publication is found here : https://doi.org/10.1093/biomethods/bpaa014. Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066. Primer sequences are here (and listed directly in the protocol) :

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