ion torrent and minion
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Ion Torrent and Minion. Relatively low cost ‘next generation’ sequencing. Wendy Smith School of Computing Science , Alan Ward Newcastle University, UK. Overview. Projects Sequencing systems Ion Torrent - introduction - PowerPoint PPT PresentationTRANSCRIPT
Ion Torrent and Minion
Wendy Smith
School of Computing Science,
Alan Ward
Newcastle University, UK
Relatively low cost ‘next generation’ sequencing
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Overview
• Projects• Sequencing systems• Ion Torrent - introduction
- steps involved in producing a sequenced genome
- costs
- results!
What is Next Generation Sequencing?
• Ion Torrent is CURRENT generation sequencing
Minion MAY be next generation?• Like Sanger, most currently available NGS systems extend a
primed template to determine a DNA sequence• Shorter reads – in 50-200bp range versus 1000bp for Sanger• BUT perform VERY LARGE numbers (million or more) of
‘short’ reads in parallel in single run to compile a database
= increased coverage – sufficiently large to cover genomes
– a high throughput approach• PacBio/Oxford Nanpore (Minion/Gridion) are single molecule
and long reads• Oxford nanopore is not sequencing by synthesis
Major players - the big three
Company Sequencing system
Illumina Illumina (= Solexa) (market leader)
ABI ‘SOLiD’
Roche ‘454’ sequencing
Advantages: Established commercially since 2008 – well tested
Limitations: COST
More affordable recent additions
Typically quarter of the cost of the ‘big three’
Company Sequencing system
Life Technologies IonTorrent - available since end of 2010
Illumina MiSeq
Oxford Nanopore Nanopore ‘trialled’ in selected labs, but only just emerging
Pacific Biosciences PacBio commercially (2012)
Various others still under development
IonTorrent – overall approach(similarities to ‘big three’)
• DNA fragments immobilised on tiny beads (called ‘Ion Sphere Particles’ or ISPs) – aiming for a SINGLE fragment per bead
• Fragments (templates) amplified by PCR – multiple copies of same template molecule on any particular bead
• Beads distributed amongst > million individual wells on a reaction ‘chip’ (aiming for one bead per well) – each well a separate ‘reaction chamber’ with separate ‘sensor’
• Detect incorporation of specific dNTPs by DNA polymerase
How does it work?
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Ion sequencing chip
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The chip: an individual well
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Overview of sequencing workflow
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Library preparation
Genomic DNA
Fragment shear reagents
Adapter compatable DNA
AdaptersAP1
Ligate Adapters and nick-repair
Size-select unamplified library(Preselected 200 or 100 base-reads)
A P1
Amplify the library
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Size select fragments
Assess the size distribution of the library using a bioanalyser
Library size 200 base read- target peak size 330bp
200 base read library gel
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Check fragment size and DNA concentration
Critical Step to determine dilution factor for template preparation and assess the size distribution of library
• Agilent Bioanalyzer with high sensitivity DNA Kit
From[bp]To [bp] % ofTotal Average Size[bp] Size distribution inCV[ %]Conc.[pg/μl] Molarity[pmol/l] 200 1,000 80 305 16.6 645.42 3,270.1
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Template preparation
Libraries now ready for the downstream Template Preparation using the Ion One touch machine:
Ion One Touch
• Adds fragments to ‘beads’ (ISPs) in proportions that yield < one fragment per bead.
• Performs Emulsion PCR to amplify the fragments on beads aim- one fragment/bead – clonal amplification
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Clonal Amplification
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Clonal Amplification
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J
L
Excluded(no target formagnetic bead)
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Load Chip with enriched particles
• Wells designed to accommodate single bead only• Require sufficient amplified ‘loaded’ beads to occupy majority of wells (confirmed by Quibt machine)• If earlier dilutions correct, should have few’mixed’ templates
Ion Torrent PGM
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Prep for every 2 runs:
• Clean (wash) PGM machine• Initialize machine and prepare solutions
For each sequencing run:
• Anneal sequencing primer• Perform polymerase binding• Load the ion Chip• Sequencing run
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The PGM Sequencing run
• As mentioned earlier, individual dNTPs are passed over the chip one at a time, with each pass followed by a wash – An individual ‘pass + wash’ is called “a flow”
• Four ‘flows’ are described as a ‘cycle’ – but every cycle is not identical – e.g. its not simply ATGC, ATGC, ATGC over and over again.
• Instead, it repeats a specific set of 8 cycles (i.e. 32 flows) with each base represented 8 times – apparently this reduces systemic errors, but they do not explain how (?)
Flows and Cycles
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Flows and Cycles
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Flows and Cycles
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Flows and Cycles
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Ionograms
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Data Analysis
Export sequence data in suitable format to ‘end-user’computer for other required analysis
Workflow – Realistic Times
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Step Parallel numbers Time
DNA preps + checks 6 – 12 ½ day
Fragment library 2 4 - 5 hr
Size selection + checks 2 2 -3 hr
Template prep 1 5 hr
Enrichment 1 1 ½ hr
PGM – Sequencing run 1 ½ day*
*Doubled if 2 chips needed to provide sufficient coverage,
Costs
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Items cost/ bact genome (£)
200bp sequencing kit (£700) 70
200 bp OneTouch Systems kit (£700) 70
BioAnalyer DNA high sensitivity kit 20
[ and/or Q-PCR kit (£700)]
E-gels 6
Sequencing chip 314 (£70) x 2 140[ or sequencing chip 316 x 1 @ > £200]
Overall – consumable costs minimum £300 – £400 per 314 chip run
Sequencing chips
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Sequencing is just the start....
• Analysis is also a bottleneck....
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Comparison to run summary from IonTorrent literature
My runManufacturer’s run report
491,020 92%
536,694 43%
9,543 2%
481,477 98%
481,477 85,212 18%
35 < 1%
61,104 13%
335,050 70%
1,262,519
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Ion Torrent de novo analysis of Mycobacterium chelonae
• Mycobacterium abscessus is an emerging pathogen• Part of the Mycobacterium chelonae clade in the fast growing
mycobacteria• Both Mycobacterium chelonae and Mycobacterium abscessus
can colonize cystic fibrosis patient’s lungs• When CF patient requires a lung transplant (late 20s early 30s)
these mycobacteria cause problems– Mycobacterium chelonae can usually be treated – Mycobacterium abscessus usually cannot
• Species are difficult to identify/distinguish
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Ion Torrent de novo analysis of Mycobacterium chelonae
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Ion Torrent de novo analysis of Mycobacterium chelonae
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de novo assembly
• CLC Workbench• Geneious• MIRA• Velvet
Very fast, very expensive
Very user friendly, cheapVery thorough, free
User unfriendly, very high memory needs, freePart of nice Virtual BoxPAGIT
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M. chelonae vs M. abscessus with r2cat
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M. chelonae vs M. abscessus with r2cat
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Alignment of de novo contigs
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Alignment using Abacas viewed in ACT
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Gap is in alignment not in assembly
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but still need another run with another ngs chemistry
• Ion Torrent run gave 80-100x coverage – possibly too much
• Different assemblers give slightly different contigs – I like MIRA for assembly – has active development and active user group – but using more than one assembler looks like a good strategy
• Need a scaffolder to assemble contigs – GAP4 recommended but I’m still getting to grips – Abacas gives detailed alignment of contigs onto reference using ACT – r2cat gives easy to use mapping onto reference genome
• but looks like have gaps • Ion Torrent library preparation is a lot of work
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MinionNext generation sequencing?
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Nanopore sensing
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Label free strand sequencing
46Glenn (2013)http://www.molecularecologist.com/next-gen-fieldguide-2013/
Comparison of ngs systems
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Minion
Advantages Disadvantages• Low capital cost (zero) High cost/Mb• Sample preparation No protocol yet• Long reads Not available!• Hairpin to read both strands• Same error rate along read 4% error rate (?)• Real time analysis Error not random• Multiplex samples
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MAP Application form
https://www.nanoporetech.com/technology/the-minion-device-a-miniaturised-sensing-system/map-application-form
Acknowledgements
Alan Ward to Wendy Smith for Ion Torrent data
Wendy Smith to:• Dr. Jen Hallinan• Prof. Anil Wipat• Dr. Graham Colin
• Prof. Colin Harwood• Dr. John Perry• Prof. Kate Gould
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