Ken McGrath - Next Gen Sequencing - Game of Thrones edition

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Title: Next‐generation sequencing: an overview of technologies and applications …

Title: Next‐generation sequencing: an overview of technologies and applications

Presenter: Dr Ken McGrath, Australian Genome Research Facility

Abstract: The “Next‐Generation Sequencing” landscape is one of constant change, with new and emerging technologies constantly competing with established platforms. This abundance of competition is resulting in faster and cheaper methods to perform sequencing of DNA and RNA samples, but it also brings with it a confusing array of options, each with its own strengths and weaknesses. Ken gives an overview of the available sequencing technologies and runs through some example projects that can be run on them, as well as describing the typical bioinformatics approaches for these projects, and also take a look at what’s “next” in Next‐Gen.

First presented at the 2014 Winter School in Mathematical and Computational Biology http://bioinformatics.org.au/ws14/program/

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  • 1. Next-Generation Sequencing: an overview of technologies and applications July 2014 Ken McGrath Australian Genome Research Facility
  • 2. Next-Gen Sequencing Edition
  • 3. • Current rulers of the “throne” • Sequencing by synthesis • Each cycle extends and reads a single base • Reads of up to 2x300bp
  • 4. DNA (0.1-1.0 ug) Sample preparation Cluster growth 5’ 5’3’ G T C A G T C A G T C A C A G T C A T C A C C T A G C G T A G T 1 2 3 7 8 94 5 6 Image acquisition Base calling T G C T A C G A T … Sequencing Illumina Sequencing Technology Robust Reversible Terminator Chemistry Foundation
  • 5. MiSeq Illumina NextSeq500HiSeq2500
  • 6. MiSeq Illumina NextSeq500HiSeq2500 GAIIx
  • 7. Illumina X Ten
  • 8. ILLUMINA SEQUENCING SYSTEMS • 150 bp paired end reads ~120Gbp / run (~1 day)NextSeq500 • 150 bp paired end reads ~ 180 Gbp/ run (2 days) Illumina HiSeq 2500 Rapid SBS • 125 bp paired end reads ~ 1000 Gbp/ run (6 day) Illumina HiSeq 2500 v4 SBS • 300 bp paired end reads ~15 Gb/run (2.3 days)MiSeq v3 • 150bp paired end reads ~1800 Gb/run (3 days)HiSeq X Ten
  • 9. ILLUMINA SEQUENCING SYSTEMS • 10 -15 million pass filter clusters per runMiSeq v2 • 50 bp single reads (0.5 – 0.75 Gb/run) ~6hrs • ≥ 90% bases higher than Q30 at 50 bp50 cycles • 150 bp paired end reads (3.0 – 4.5 Gb/run) ~24 hrs • ≥ 80% bases higher than Q30 at 2x150 bp300 cycles • 2x250 bp paired end reads (5.0 - 7.5 Gb/run) ~40 hrs • ≥75% bases higher than Q30 at 2 x 250 bp500 cycles • 20-25 million pass filter clusters per runMiSeq v3 • 2x 75 bp paired end reads (3.0 – 2.5 Gb/run) ~20 hrs • ≥ 85% bases higher than Q30 at 2 x 75 bp150 cycles • 2x300 bp paired end reads (12.0 – 15.0 Gb/run) ~55 hrs • ≥ 70 % bases higher than Q30 at 2 x 300 bp600 cycles
  • 10. Illumina Summary Strengths Weaknesses Lots of data  Too much data  Low error rates Slower run times Great choice of platform sizes Shorter reads Paired-end reads Pretty awesome Slept with brother
  • 11. • Competing with illumina for market share • Two technologies (sequencing by ligation, and semiconductor sequencing) • Reads of up to 400bp
  • 12. Ion Torrent • Ion Semiconductor Sequencing •Detection of hydrogen ions during the polymerization DNA • Sequencing occurs in microwells with ion (pH) sensors – No modified nucleotides – No optics
  • 13. Ion Torrent • DNA Ions  Sequence – Nucleotides flow sequentially over Ion semiconductor chip – One sensor per well per sequencing reaction – Direct detection of natural DNA extension – Millions of sequencing reactions per chip – Fast cycle time, real time detection Sensor Plate Silicon Substrate Drain SourceBulk dNTP To column receiver ∆ pH ∆ Q ∆ V Sensing Layer H+
  • 14. SOLiD Life Technologies Ion Torrent PGM Ion Torrent Proton
  • 15. •100 bp reads ~20 Gbp/run (Coming soon!) Ion Torrent Chips •200bp and 400bp reads, 30-100Mb/run (1.5 hrs) 314 Chip •200bp and 400bp reads, 300-1000 Mbp / run (2 hrs)316 Chip •200bp and 400bp reads, 600Mb-2Gbp / run (4.5 hrs)318 Chip •200 bp reads, 5-10 Gbp/runP1 Chip P2 Chip PGMPROTON
  • 16. Life Technologies Summary Strengths Weaknesses Fast run times Lower maximum data output Scalable data outputs Read quality can vary Longer reads (400bp) Pretty Haven’t done much recently
  • 17. • Current rulers of the throne • Sequencing by synthesis • Each cycle extends and reads a single base • Reads up to 2x300bp
  • 18. • Current rulers of the throne • Sequencing by synthesis • Each cycle extends and reads a single base • Reads up to 2x300bp • One of the first NGS platforms • Pyrosequencing based • Each cycle allows extension of a single base (A, C, G or T) • Reads up to 800bp
  • 19. 454 Pyrosequencing
  • 20. 454 Pyrosequencing
  • 21. 454: Data Processing Image Processing Base- calling Quality Filtering SFF File T Base Flow A Base Flow C Base Flow G Base Flow Raw Image Files
  • 22. GS-FLX Roche FLX Jr
  • 23. GS-FLX Roche FLX Jr
  • 24. Roche • Not over yet… Stratos GenomicsGenia Something else?
  • 25. Roche Summary Strengths Weaknesses Long reads (up to 800bp) High $ per base Older technology Platform soon unavailable Had wolves Pretty much dead
  • 26. • Competing with illumina for market share • Two technologies (sequencing by ligation, and semiconductor sequencing) • Reads of up to 400bp
  • 27. • Competing with illumina for market share • Two technologies (sequencing by ligation, and semiconductor sequencing) • Reads of up to 400bp • Single-molecule real-time sequencing (SMRT) • Detection of individual bases as they extend (by light emission) • Long Reads (up to 4x2.5kb)
  • 28. PacBio
  • 29. PacBio • Higher error rates (~90%) • Compensate by “looping” DNA to create multiple passes
  • 30. PacBio Zero-Mode Waveguides (ZMW)
  • 31. PacBio Summary Strengths Weaknesses Long reads (4x2.5kb) High $ per base Single-molecule detection Higher error rate Capable of Epigenetics Still to prove itself Freakin’ Dragons! Keeps losing dragons
  • 32. Oxford Nanopore
  • 33. • Direct detection of individual bases as pass through a “nanopore” • MinION and GridION • No synthesis/extension • Capable of VERY Long Reads (>100kb)
  • 34. Oxford Nanopore Summary Strengths Weaknesses Extra-Long reads (>100kb) Not yet available (alpha testing) Single-molecule detection Very high error rates Capable of Epigenetics Immature platform Very cost effective Exotic and powerful Steal babies
  • 35. NGS Applications • Whole genome sequencing (today) » De novo assembly » Structural variant detection » Comparative genomics • RNAseq (later today) » Gene expression » Splice variants » Transcriptomics » MicroRNA • Epigenomics (tomorrow) » Indirect (bisulphite) » Direct • Targeted sequencing (Wed) » Hybrid capture » Amplicon resequencing Data Quality Read Length Yield/ Coverage
  • 36. Hodor! (Thank You)