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Molecular Typing of Hepatitis B Using Fusion Primers and IonTorrent
Author(s) -
Ringlander Johan,
Andersson Maria,
Lindh Magnus
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb557
Subject(s) - amplicon , hepatitis b virus , sanger sequencing , genome , biology , dna sequencing , virology , genotyping , primer (cosmetics) , massive parallel sequencing , hepatitis b , nanopore sequencing , genetics , computational biology , polymerase chain reaction , genotype , virus , dna , gene , chemistry , organic chemistry
Background Hepatitis B virus (HBV) chronically infects 250 million people, and is the main contributor to hepatoceullular carcinoma (HCC) in the world. When contracted at adult age, HBV can cause acute hepatitis which sometimes is severe and may result in acute liver failure. Molecular epidemiology is important for the analysis of circulating HBV strains and for clarifying transmission chains. Next‐generation sequencing (NGS) has several advantages over traditional Sanger sequencing, but high costs for NGS, in particular the library preparation step, has restricted its use. Objective To develop a cost‐efficient strategy for simplified amplicon sequencing of HBV whole genomes on IonTorrent S5 (Thermofischer, Watlham, MA, USA), and an automated bioinformatics pipeline. Method Primers with tails carrying IonTorrent A or P1 adaptor, were designed to target conserved regions using an alignment consisting of fifty HBV strains, reflecting all known genotypes. The primers were located to cover the genome by 10 amplicons of approximately 380 bp size, and will be presented. HBV genomes in serum samples was amplified by duplex PCR in five parallel reactions, followed by sequencing in IonTorrent S5, and bioinformatic processing by CLC Genomics Workbench (Qiagen, Aarhus, Denmark). Results All ten overlapping segments of HBV DNA from four patient sera, representing genotypes A, B, C and D were successfully amplified. Sequencing data are yet to be analyzed and will be presented. Discussion and conclusion To our knowledge, whole genome sequencing of HBV using fusion primer derived amplicons for IonTorrent have not previously been published. By this technique, whole genome sequencing of HBV can be performed in large scale and at low cost. Support or Funding Information Governmental strategic research funds, Sahlgrenska University Hospital. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .