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

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Out

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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

17

Partial Order Graphs

maximum weight path GCTCGAT is the corrected read

Error Correction

Contig Assembly

20

Celera Assembler produces one contig at 98.5% identity

Selecting a Consensus

21

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/