lab in a suitcase and other adventures with nanopore sequencing
TRANSCRIPT
Lab in a Suitcase and Other Adventures with Nanopore Sequencing
Josh QuickUniversity of Birmingham
Oxford Nanopore MinION
Unique properties:• Real-time• Portable• Long reads
Real-time sequencing
PHE
Surv
eilla
nce
Out
brea
k
Library prep time: 92 minutes
Moving to real-time sequencing ….
1x
2x
Serogroup IDAlign MinION reads to S.enterica reference genome. Phylogenetic placement with pplacer
Within 40 mins we could identify the strains as serotype Enteritidis
Outbreak IDAlignment 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
De novo assembly
Nanopore assembly pipeline
Error correction
Celera Assembler
Consensus
Input reads
Genome Assembly
Input Data• First challenge is finding overlaps for reads with 15-20% errors
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Partial Order Graphs
maximum weight path GCTCGAT is the corrected read
Error Correction
Contig Assembly
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Celera Assembler produces one contig at 98.5% identity
Selecting a Consensus
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Mutate
ACTACGATCGACTTACGACCTACGATCGACTTACGATCTACGATCGACTTACGA
...-CTACGATCGACTTACGAG-TACGATCGACTTACGAGC-ACGATCGACTTACGAGCT-CGATCGACTTACGA
...GACTACGATCGACTTACGAGCCTACGATCGACTTACGAGGCTACGATCGACTTACGAGTCTACGATCGACTTACGA
-190-192-171 -176-191-193-168 -198-191-195-181
GCTACGATCGACTTACGA
Assembly AccuracyDraft: 98.5% accuracy Polished: 99.5% accuracy
Assembly Accuracy
3khz / MAP-004 update
Ebola
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
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
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%
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
0.0001
microreact.org Mixing between Kambia and Forecariah casesGuinea ‘lineage A’ present in SL
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/
AcknowledgementsBirmingham• 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/