RT Journal Article
SR Electronic
T1 Extraction-free direct PCR from dried serum spots permits HBV genotyping and RAS identification by Sanger and minION sequencing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 552539
DO 10.1101/552539
A1 Stuart Astbury
A1 Marcia Maria Da Costa Nunes Soares
A1 Emmanuel Peprah
A1 Barnabas King
A1 Ana Carolina Gomes Jardim
A1 Jacqueline Farinha Shimizu
A1 Paywast Jalal
A1 Chiman H Saeed
A1 Furat T Sabeer
A1 William L Irving
A1 Alexander W Tarr
A1 C Patrick McClure
YR 2019
UL http://biorxiv.org/content/early/2019/02/16/552539.abstract
AB In order to achieve the commitment made by the World Health Organisation to eliminate viral hepatitis by 2030, it is essential that clinicians can obtain basic sequencing data for hepatitis B virus (HBV) infected patients. While accurate diagnosis of HBV is achievable in most clinical settings, genotyping and identification of resistance-associated substitutions (RAS) present a practical challenge in regions with limited healthcare and biotechnology infrastructure. Here we outline two workflows for generating clinically relevant HBV sequence data directly from dried serum spot (DSS) cards without DNA extraction using either Sanger, or the portable MinION sequencing platforms. Data obtained from the two platforms were highly consistent and allowed determination of HBV genotype and RAS. This is the first demonstration of MinION sequencing from DSS, illustrating the broad utility of this sequencing technology. We demonstrated the clinical application of this technology using sera sampled on DSS cards obtained from both Iraq and Brazil. The sample stability provided by DSS cards, combined with the rapid PCR and sequencing protocols will enable regional/national centres to provide information relevant to patient management. By providing viable workflows for both the Sanger and MinION sequencing platforms, which vary greatly in the infrastructure and expertise required, we demonstrate that MinION sequencing is a viable method for HBV genotyping in resource-limited settings. These workflows could also be applied to sequencing of other blood borne DNA viruses and bacterial pathogens.