single-molecule real-time (smrt) nanopore sequencing for plant pathology applications
TRANSCRIPT
Single-molecule real-time (SMRT) Nanopore sequencing for Plant Pathology applications
Joe ParkerEarly-career Research Fellow (Phylogenomics), Royal Botanic Gardens, KewSkype, Twitter, Github: @lonelyjoeparkerEmail: [email protected]
SMRT / Nanopore sequencing for Plant Pathology
Topics:• What is it?• Unique capabilities• Current work• Plant health
applications
What is Nanopore sequencing?
Unique capabilities
• Portable• Real-time (~minutes)• DNA and RNA• Selective (‘read-until’)
sequencing• Very long reads• Flexible / scalable
Real projects (i)
Real-time, portable sequencing for ebolavirus (EBV) surveillance, 2015 (Guinea)
Local sequencing in primary care, limitations on:
• facilities, • internet bandwidth,• computation
Data uploaded for further analysis worldwide
Sampling-to-answer in ~daysPhylogeography, demography and evolution of EBV monitored in real-time
Quick et al. (2016)http://dx.doi.org/10.1038/nature16996
Real projects (ii)
Field-based genomics for plants:
a) Unsupported sequencing and real-time identification of Arabidopsis spp. in Snowdonia
Complete leaf-to-analysis workflow in the field
Field-sequenced data comparable to lab
b) Real-time identification of randomised samples
Six diverse plant species randomised and sequenced in tent at Kew Science Festival
Identification < 1hour Minimal reference needed
Papadopulos & Parker (in prep.)
Real projects (iii)
Real-time selective sequencing on nanopore technology• Proportion of desired sequences from WGS is low• SMRT is a real-time platform, why not pick and choose individual DNA molecules?• Phage lambda regions selectively sequenced with software-based method• Any region could be selected for resequencing, and alterations to protocol made during a run
Loose et al. (2016)http://dx.doi.org/10.1038/nmeth.3930
Real projects (iv)
RNA sequencing:
-RNA sequenced directly from sample-Stochiometric / quantitative-Tested on E.coli Major implications for transcriptomics, RNA virus detection
Garalde et al. (submitted)http://dx.doi.org/10.1101/068809
GCPP Applications
• Cryptic parasite / pathogen spp. discovery
• Real-time monitoring of plant health
• Import / export sequencing
Thanks
Collaborators / funders: – Kew Foundation Pilot Study Fund– Drs. Dion Devey, Andrew Helmstetter, Alexander S.T. Papadopulos– Oxford Nanopore Technologies– Snowdonia NPA– Natural Resources Wales
Contact:Skype, Twitter, Github: @lonelyjoeparkerEmail: [email protected]