Poster Presentation Australian Society for Medical Research Annual Scientific Meeting 2016

Evaulatiton of a novel nanopore method for whole genome sequencing of an RNA virus (#121)

Ross D Balch 1 2 , Kirsten Spann 1 2 , Seweryn Bialasiewicz 2 3
  1. Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
  2. Centre for Children's Health Research, Children's Health Queensland, South Brisbane, Queensland, Australia
  3. Queensland Paediatric Infectious Diseases Laboratory, Children's Health Queensland, South Brisbane, Queensland, Australia

Introduction: MinION nanopore sequencing is a novel NGS method that requires minimal sample input and library preparation with a run cost of less than $1000. The device is small and portable which makes it ideal for bench top or field work. Clinical use of such a method would enable rapid field epidemiology and cheaper routine surveillance.


Objectives: In this study we seek to validate the nanopore method of sequencing for routine use in the laboratory to analyse clinical samples of RNA respiratory viruses with respect to sequencing error rate and workflow considerations.


Methodology: RNA will be extracted from viruses derived from cell culture supernatant. RT-PCR will be performed producing 9 overlapping sequence fragments ~2 kbp in length. The PCR products will be pooled and each preparation will then undergo library preparation as per the manufacturer’s instructions and subject to nanopore sequencing on the MinION. The fusion (F) and the nucleocapsid (N) gene will be subject to PCR producing 4 overlapping fragments per gene ~ 800 bp in length to be used for Sanger sequencing. The data from the nanopore method will be compared to the published sequence of the established RSV strain as well as the Sanger sequence to determine if the nanopore method is suitable for routine laboratory use.   


Expected Results: It is expected that sequence will be generated of the whole viral genome at a sequence depth of at least 25 – 50 reads/bp. The error rate per base per read as well as the error rate per base in a consensus sequence will be determined. Workflow time and difficulty of execution will also be determined


Conclusions: It is expected that the sequence will be of sufficient quality to use in routine diagnostic and epidemiologic analysis in both a clinical and research setting.