Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples

Abstract

Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.

Characterizing Phage Genomes for Therapeutic Applications

“…Short read technologies typically (1) have inherently low error rates and (2) due to the high throughput, resulting deep coverage [ 59 ]. On the contrary, long read sequencing platforms such as the Oxford MinION and PacBio hold promise for resolving terminal repeats and other ambiguities in phage genome assemblies, but the combination of higher error rates and less deep coverage [ 60 , 61 , 62 ] is likely to make error correction with short reads a necessity in many cases for at least the near future. The pipeline presented in this manuscript would require adjustment to be suitable for long reads, particularly the QC and assembly steps, and subsampling would likely not be required for long reads.…”

Direct sequencing of RNA with MinION Nanopore: detecting mutations based on associations

“…Longer indels may also occur (2), as may events of genetic recombination. Disease pathogenesis, progression, management and epidemiology are all affected by the genetic diversity created in microbial populations (3)(4)(5)(6)(7)(8)(9). Thus, characterizing this diversity is of utmost importance in clinical as well as research settings, which makes the development and improvement of suitable sequencing technologies crucial (10)(11)(12).…”

Nanopore‐Based, Rapid Characterization of Individual Amyloid Particles in Solution: Concepts, Challenges, and Prospects

“…Some of these benefits, such as the characterization of shapes, volumes, diffusion coefficients, and electrical and mechanical properties of individual proteins and protein complexes, are appealing for fundamental biophysical studies. Other advantages, including the ability to extract resistive pulses from single unlabeled molecules and to perform analyses of those resistive pulses in real time, may be clinically useful . This article focuses specifically on the application of resistive pulse sensing to amyloid‐related protein analytes and discusses the concepts and challenges of this application.…”