Lab in a Suitcase and Other Adventures with Nanopore Sequencing
- 1. Lab in a Suitcase and Other Adventures with Nanopore Sequencing Josh Quick University of Birmingham
- 2. Oxford Nanopore MinION Unique properties: • Real-time • Portable • Long reads
- 10. Library prep time: 92 minutes
- 11. Moving to real-time sequencing …. 1x 2x
- 13. Serogroup ID Align MinION reads to S.enterica reference genome. Phylogenetic placement with pplacer Within 40 mins we could identify the strains as serotype Enteritidis
- 14. Outbreak ID Alignment of MinION reads to Salmonella Enteritidis reference genome. Phylogenetic placement with pplacer Within 100 minutes the outbreak strains was unambiguously part of the national 14b cluster (RED) and the sporadic cases (BLUE) was indeed sporadic Quick et al – Genome Biology
- 15. De novo assembly
- 16. Nanopore assembly pipeline Error correction Celera Assembler Consensus Input reads Genome Assembly
- 17. Input Data • First challenge is finding overlaps for reads with 15-20% errors 1
- 18. Partial Order Graphs maximum weight path GCTCGAT is the corrected read
- 19. Error Correction
- 20. Contig Assembly 2 Celera Assembler produces one contig at 98.5% identity
- 22. Assembly Accuracy Draft: 98.5% accuracy Polished: 99.5% accuracy
- 24. 3khz / MAP-004 update
- 25. Ebola
- 26. DSTL Porton Down Protocol Development • Two approaches tried on archived Ebola RNA: – Genome tiling RT-PCR amplicons – Direct metagenomics: total RNA sequencing of Ebola- spiked sample • Validation with MiSeq
- 35. Guinea 11 reactions v2, 98.4% coverage Guinea 19 reactions v1, 98.1% coverage Guinea 11 reactions v1, 95.9% coverage Porton Down validation set, 89.1% coverage
- 36. Sample Scheme Transferred reads Mean coverage Aligned 2D passing Mismatch rate 014370 19rx 10,000 372x 6,929 (69%) 9% 015802 11rx 10,000 345x 4,200 (42%) 11.7% 004674 11rx 5,000 219x 2,647 (52%) 10.1% 015815 11rx 10,000 347x 4,256 (42%) 10.2% 015972 11rx V2 5,000 178x 2,038 (40%) 8.7% 015986 11rx V2 5,000 211x 2,438 (48%) 9.1%
- 38. RT-PCR Quantification and pooling Library preparation MinION sequencing Upload data Basecalling MarginAlign MarginCaller Manual inspection Consensus ML tree 3 hours 1 hour 2 hours 15-60m 30-120m 20m 1 hour Jain et al. – Nature Methods
- 46. microreact.org Mixing between Kambia and Forecariah cases Guinea ‘lineage A’ present in SL
- 48. Bioinformatics opportunities • Squiggle space alignment without base calling • Variant calling from squiggles • Run-until for pool balancing • HMM can also align events to a reference genome • – Read about it here: • http://simpsonlab.github.io/2015/04/08/eventalign/
- 49. Acknowledgements Birmingham • Nick Loman OICR • Jared Simpson DSTL Porton Down • Simon Weller • Jamie Taylor • Phil Rachwal • Carl Mayers Public Health England • Miles Carroll EMLab • Sophie Daffour • Martin Gabriel • Stefan Gunther Edinburgh • Andrew Rambaut Liverpool • Julian Hiscox • Georgios Pollakis Bristol • Dave Matthews Oxford Nanopore • Lots http://ebola.nextflu.org /