Amplicon-based virus sequencing
The general approach to this protocol is to amplify the virus genome in small (~400 bp) overlapping fragments using two highly multiplexed PCR reactions (where the overlapping segments are in separate reactions). Think about it as the ‘jackhammer’ approach. It great for all sample types, but especially useful if they are degraded or low titer. You can use one of our primer schemes or you can design a scheme of your own. The protocol is easy, cheap, and scalable.
Methods for using our protocol to measure intrahost diversity and analysis software are also available.
Paper: clinical sequencing Paper: intrahost analysisMiSeq protocol for virus sequencing
Originally developed for Zika virus, our simple and scalable amplicon-based sequencing approach, PrimalSeq, can be used for a variety of viruses.
protocolMinION protocol for virus sequencing
Sequencing viruses in the field? Try the amplicon-based PrimalSeq approach adapted for use on the hand-held MinION.
protocolPrimer schemes for amplicon sequencing
We currently have validated schemes to sequence the full coding sequence of Zika virus, chikungunya virus, yellow fever virus, dengue virus 2, Usutu, and West Nile virus.
primersCreate your own primer schemes
Sequencing a different virus? Our amplicon-based approach can be adapted to a variety of viruses by designing your own primers using this handing design tool, Primal Scheme.
websiteIllumina data analysis
Open access software, iVar, to generate consensus sequences and identify intrahost variants from Illumina amplicon data.
softwareMinION data analysis
Open access methods to process and generate consensus sequences from MinION amplicon data.
pipelineContributors
University of Birmingham
PrimalScheme, many of the amplicon sets, and the MinION protocols were developed by Nick Loman and Josh Quick.
Loman LabScripps Research Institute
The Illumina protocols, iVar, and many of the amplicon sets were developed by Kristian Andersen, Karthik Gangavaru, and Glenn Oliveira.
Andersen LabZiBRA Project
Implimentation of field-deployable protocols and analysis were developed by the Zika in Brazil Real-time Analysis team.
ZiBRAArboSPREAD
Be on the lookout for new amplicon sets and data to track the spread of arboviruses in Africa and South America led by Nuno Faria
Faria Lab