ion torrent and minion

Ion Torrent and Minion

Wendy SmithSchool of Computing Science,

Alan WardNewcastle 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 testedLimitations: COST

More affordable recent additions

Typically quarter of the cost of the ‘big three’Company Sequencing systemLife Technologies IonTorrent - available since end of 2010Illumina 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

Adapters AP1

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 TimeDNA 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) 70BioAnalyer DNA high sensitivity kit 20 [ and/or Q-PCR kit (£700)]

E-gels 6Sequencing 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 expensiveVery user friendly, cheapVery thorough, freeUser 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 dataWendy Smith to:• Dr. Jen Hallinan• Prof. Anil Wipat• Dr. Graham Colin

• Prof. Colin Harwood• Dr. John Perry• Prof. Kate Gould

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