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Ion Torrent and Minion

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

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Ion Torrent and Minion

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  1. Ion Torrent and Minion Relatively low cost ‘next generation’ sequencing Wendy Smith School of Computing Science, Alan Ward Newcastle University, UK

  2. Overview • Projects • Sequencing systems • Ion Torrent - introduction - steps involved in producing a sequenced genome - costs - results!

  3. 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

  4. Major players - the big three Company Sequencing system IlluminaIllumina (= Solexa) (market leader) ABI ‘SOLiD’ Roche ‘454’ sequencing Advantages: Established commercially since 2008 – well tested Limitations: COST

  5. More affordable recent additions Typically quarter of the cost of the ‘big three’ Company Sequencing system Life Technologies IonTorrent - available since end of 2010 IlluminaMiSeq Oxford Nanopore Nanopore ‘trialled’ in selected labs, but only just emerging Pacific Biosciences PacBio commercially (2012) Various others still under development

  6. 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

  7. How does it work?

  8. Ion sequencing chip

  9. The chip: an individual well

  10. Overview of sequencing workflow

  11. Library preparation Genomic DNA Fragment shear reagents Adapter compatable DNA Ligate Adapters and nick-repair A P1 Adapters Size-select unamplified library (Preselected 200 or 100 base-reads) A P1 Amplify the library

  12. Size select fragments Library size 200 base read- target peak size 330bp 200 base read library gel Assess the size distribution of the library using a bioanalyser

  13. 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

  14. Template preparation Libraries now ready for the downstream Template Preparation using the Ion One touch machine: • Adds fragments to ‘beads’ (ISPs) • in proportions that yield < one • fragment per bead. Ion One Touch • Performs Emulsion PCR to • amplify the fragments on beads • aim- one fragment/bead – clonal amplification

  15. Clonal Amplification

  16. Clonal Amplification

  17. J L Excluded (no target for magnetic bead)

  18. 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

  19. Ion Torrent PGM 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

  20. 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 (?)

  21. Flows and Cycles

  22. Flows and Cycles

  23. Flows and Cycles

  24. Flows and Cycles

  25. Ionograms

  26. Data Analysis Export sequence data in suitable format to ‘end-user’ computer for other required analysis

  27. Workflow – Realistic Times 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,

  28. Costs Items cost/ bact genome (£) 200bp sequencing kit (£700) 70 200 bpOneTouch 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

  29. Sequencing chips

  30. Sequencing is just the start.... • Analysis is also a bottleneck....

  31. Comparison to run summary from IonTorrent literature Manufacturer’s run report My run 1,262,519 536,694 43% 491,020 92% 9,543 2% 481,477 98% 481,477 85,212 18% 35 < 1% 61,104 13% 335,050 70%

  32. Ion Torrent de novo analysis of Mycobacterium chelonae • Mycobacterium abscessusis an emerging pathogen • Part of the Mycobacterium chelonaeclade in the fast growing mycobacteria • Both Mycobacterium chelonaeand Mycobacterium abscessuscan colonize cystic fibrosis patient’s lungs • When CF patient requires a lung transplant (late 20s early 30s) these mycobacteria cause problems • Mycobacterium chelonaecan usually be treated • Mycobacterium abscessususually cannot • Species are difficult to identify/distinguish

  33. Ion Torrent de novo analysis of Mycobacterium chelonae

  34. Ion Torrent de novo analysis of Mycobacterium chelonae

  35. de novo assembly • CLC Workbench • Geneious • MIRA • Velvet Very fast, very expensive Very user friendly, cheap Very thorough, free User unfriendly, very high memory needs, free Part of nice Virtual Box PAGIT

  36. M. chelonaevsM. abscessuswith r2cat

  37. M. chelonaevsM. abscessuswith r2cat

  38. Alignment of de novo contigs

  39. Alignment using Abacas viewed in ACT

  40. Gap is in alignment not in assembly

  41. 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

  42. MinionNext generation sequencing?

  43. Nanopore sensing

  44. Label free strand sequencing

  45. Comparison of ngs systems Glenn (2013) http://www.molecularecologist.com/next-gen-fieldguide-2013/

  46. Minion AdvantagesDisadvantages • 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

  47. MAP Application form https://www.nanoporetech.com/technology/the-minion-device-a-miniaturised-sensing-system/map-application-form

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