NGS - Basic principles and sequencing platforms
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Presentation on the basic principles of Next Generation Sequencing, delivered at the Belgian Scientific Institute of Public Health on Oct 10, 2014.
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NGS - Basic principles and sequencing platforms
- 1. Next-generation-sequencing Basic principles and sequencing platforms WIV-ISP workshop 10/10/2014 Annelies Haegeman Institute for Agricultural and Fisheries Research Plant Sciences Unit www.ilvo.vlaanderen.be Agriculture and Fisheries Policy Area
- 2. 10/10/2014 What is next generation sequencing? 2 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Ion Torrent • Illumina • 3rd generation sequencing (2012 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO
- 3. What is next generation sequencing? 3 NGS experiment setup DNA sample NGS Instrument Data Library Preparation Sequencing Data Analysis 10/10/2014
- 4. What is next generation sequencing? 4 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Ion Torrent • Illumina • 3rd generation sequencing (2012 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO 10/10/2014
- 5. What is next generation sequencing? 5 Sanger sequencing (1977) 10/10/2014
- 6. What is next generation sequencing? 6 Sanger sequencing 10/10/2014 http://en.wikipedia.org/wiki/Sanger_sequencing
- 7. What is next generation sequencing? 7 Applied Biosystems • 1987 : First sequencer (ABI370) • 1996 : First capillary sequencer (ABI 310) • Present : ABI 3500 10/10/2014
- 8. What is next generation sequencing? 8 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Ion Torrent • Illumina • 3rd generation sequencing (2012 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO 10/10/2014
- 9. What is next generation sequencing? 9 454 sequencing • 2005 • DNA is fragmented, adapter ligated and denatured • Emulsion PCR 10/10/2014 Rothberg, J.M. & Leamon, J.H. Nature Biotechnology 26, 1117 - 1124
- 10. What is next generation sequencing? 10 454 sequencing • Put beads and reagents in picotiter plate (one DNA bead per well) 10/10/2014
- 11. What is next generation sequencing? 11 454 sequencing • Sequencing • One bead = one read • Reads up to 700 bp • 1 million reads / run 10/10/2014
- 12. What is next generation sequencing? 12 454 sequencing GS Junior • ~ 400 bp reads • Up to 100.000 reads / run • 10h run time GS FLX Titanium XL+ • ~ 700 bp reads • Up to 1 million reads/run • 23h run time 10/10/2014
- 13. What is next generation sequencing? 13 454 sequencing • Pros • First commercially available NGS technique • Relatively long reads • Relatively short run time • Cons • Accuracy problems with homopolymeric repeats • Emulsion PCR • Relatively few reads • Relatively expensive • R.I.P. 2016 10/10/2014
- 14. What is next generation sequencing? 14 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Ion Torrent • Illumina • 3rd generation sequencing (2012 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO 10/10/2014
- 15. • 2010, Life Technologies • The first steps are similar to 454 sequencing • Bind DNA to beads • Emulsion PCR • Chip with ion sensor What is next generation sequencing? 15 Ion Torrent sequencing 10/10/2014
- 16. What is next generation sequencing? 16 Ion Torrent sequencing • Semiconductor sequencing 10/10/2014
- 17. What is next generation sequencing? 17 Ion Torrent sequencing • Semiconductor sequencing on a chip (no imaging) 10/10/2014
- 18. What is next generation sequencing? 18 Ion Torrent sequencing Ion PGM • 3 types of chips • 200 or 400 bp reads • Up to 5.5 million reads / Ion 318 chip • 4 – 7 h run time Ion Proton • Up to 200 bp reads • Up to 60-80 million reads • 2 – 4 h run time 10/10/2014
- 19. What is next generation sequencing? 19 Ion Torrent sequencing • Pros • Fast • Relatively cheap • Scalable (different chips) • Cons • Relatively high error rate • Emulsion PCR • Ion isothermal amplification chemistry (November 2014) • isothermal amplification instead of emulsion PCR 10/10/2014
- 20. What is next generation sequencing? 20 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Ion Torrent • Illumina • 3rd generation sequencing (2012 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO 10/10/2014
- 21. What is next generation sequencing? 21 Illumina sequencing • 2006 (Solexa) • Most popular sequencing technique nowadays! 0 2000 4000 6000 8000 10000 12000 454 solid ion torrent illumina pacbio # studies in NCBI SRA database (01/2014) 10/10/2014
- 22. 10/10/2014 What is next generation sequencing? 22 Illumina sequencing Metzker, Nature Reviews Genetics, 2010, 11, 31-46
- 23. What is next generation sequencing? 23 Paired end sequencing 10/10/2014 ©Illumina
- 24. What is next generation sequencing? 24 Illumina sequencing MiSeq • Up to 2 x 300 bp reads • 15 million single reads • 2.7 days run time HiSeq2500 • Up to 2 x 125 bp reads • 2 billion single reads • 6 days run time 10/10/2014
- 25. What is next generation sequencing? 25 Illumina sequencing NextSeq 500 • Up to 2 x 150bp reads • Up to 400 million reads • 1.2 days run time HiSeq X Ten • 2 x 150 bp reads • Up to 30 billion reads • 3 days run time 10/10/2014
- 26. What is next generation sequencing? 26 Illumina sequencing • Pros • Very high throughput • Best price/bp • Cons • Relatively long run time • Sequence quality decreases towards the end • Imaging interference in low diversity libraries 10/10/2014
- 27. What is next generation sequencing? 27 Advantages of NGS vs Sanger • Cheap (cost per bp) • 1 bacterial genome (100x, 650Mb) < a 96-well plate (80 kb) • (Very) high throughput (parallel sequencing) • Multiplexing 10/10/2014 ©Illumina
- 28. What is next generation sequencing? 28 How do I choose a platform? • Read length vs. number of reads (read depth) 10/10/2014
- 29. What is next generation sequencing? 29 Overview 10/10/2014
- 30. What is next generation sequencing? 30 Overview Platform Chemistry Read Length Run Time Reads/Run Advantage Disadvantage 454 GS Junior Pyro- sequencing 400 8 h 0.1 M Long read length High error rate, expensive 454 GS FLX+ Pyro- sequencing 700 23 h 1 M Long read length High error rate, expensive Ion PGM Proton Detection 200-400 4-7 h 5.5 M Short run time High error rate, but will improve Ion Proton Proton Detection 200 2-4 h 80 M Short run time High error rate, but will improve Illumina MiSeq Bridge amplification up to 2x300 2.7 days 25 M High- throughput / cost Long run time Illumina HiSeq Bridge amplification 2x125 6 days 2000 M High- throughput / cost Short reads, long run time 10/10/2014
- 31. What is next generation sequencing? 31 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Illumina • Ion Torrent • 3rd generation sequencing (2011 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO PCR based Single molecule 2nd 10/10/2014
- 32. What is next generation sequencing? 32 PacBio sequencing • 2011, Pacific Biosciences • Single Molecule Real Time Sequencing (SMRT) 10/10/2014
- 33. What is next generation sequencing? 33 PacBio sequencing PacBio RSII • Up to 30 kb reads • 50.000 reads • Up to 3h run time Often used in combination with Illumina sequencing 10/10/2014
- 34. What is next generation sequencing? 34 PacBio sequencing • Pros • No PCR • Very long reads • No problems with high GC content • Can detect nucleotide modifications (e.g. methylation) • Cons • High error rate, but expected to improve 10/10/2014
- 35. What is next generation sequencing? 35 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Illumina • Ion Torrent • 3rd generation sequencing (2011 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO PCR based Single molecule 10/10/2014
- 36. What is next generation sequencing? 36 Oxford nanopore sequencing 10/10/2014
- 37. What is next generation sequencing? 37 Oxford nanopore sequencing 10/10/2014 • MinION portable sequencer • Currently a beta version is being tested in 500 selected labs • Accuracy: 60-85% • First reports on read lengths: up to 79000 bases! • Under development • GridION • PromethION
- 38. 10/10/2014 What is next generation sequencing? 38 Presentation outline • Sanger sequencing (1977 – present) • Next generation sequencing (2005 – present) • 454 • Ion Torrent • Illumina • 3rd generation sequencing (2012 – present) • PacBio or SMRT sequencing • Oxford Nanopore • Sequencing @ ILVO
- 39. Animal Sciences Cattle, Pig and Small Animal Husbandry Fisheries and Aquatic Production Marine Environment and Quality Plant Sciences Crop Protection Growth and Development Applied Genetics and Breeding Crop Husbandry Technology and Food Science Agricultural Engineering Product Quality and Innovation Food Safety Social Sciences Agricultural and Farm Development Rural Development ILVO What is next generation sequencing? 3910/10/2014
- 40. Plant Sciences Crop Protection Growth and Development Applied Genetics and Breeding Crop Husbandry Sequencing @ ILVO What is next generation sequencing? 4010/10/2014 Whole genome shotgun (WGS) sequencing Plant pathogens, mainly bacteria Amplicon sequencing (16S rRNA) Bacterial populations in soil Transcriptome sequencing Azalea, perennial ryegrass Targeted resequencing Perennial ryegrass Genotyping-by-sequencing (GBS) Perennial ryegrass, Phytophthora oomycetes
- 41. Sequencing @ ILVO What is next generation sequencing? 4110/10/2014 Animal Sciences Cattle, Pig and Small Animal Husbandry Marine Environment and Quality Fisheries and Aquatic Production Amplicon sequencing (16S rRNA) Bacterial populations in: - Cow rumen - Marine environments (water, sediment, litter) Whole genome shotgun sequencing (metagenomics) - Pig gut (antibiotics resistance genes) - Marine environments (plastic litter) Genotyping-By-Sequencing (GBS) Comb jelly
- 42. What is next generation sequencing? 42 Sequencing @ ILVO www.ilvogenomics.be 10/10/2014
- 43. Questions? WIV-ISP workshop 10/10/2014 Institute for Agricultural and Fisheries Research Plant Sciences Unit www.ilvo.vlaanderen.be Agriculture and Fisheries Policy Area annelies.haegeman@ilvo.vlaanderen.be