icar soybean indore 2014

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Presented on June 7 at Directorate of Soybean Research Khandwa Road, Indore, M.P, India - 452 001 http://www.nrcsoya.nic.in/default.htm

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  • 1. Surya Saha Cornell University & Boyce Thompson Institute suryasaha@cornell.edu @SahaSurya Directorate of Soybean Research, Indore June 7,2014 Slides: http://bit.ly/Soybean_Indore_2014 http://www.acgt.me/blog/2014/3/7/next-generation-sequencing-must-die
  • 2. 6/6/2014 Directorate of Soybean Research, Indore 2 You are free to: Copy, share, adapt, or re-mix; Photograph, film, or broadcast; Blog, live-blog, or post video of; This presentation. Provided that: You attribute the work to its author and respect the rights and licenses associated with its components. Slide Concept by Cameron Neylon, who has waived all copyright and related or neighbouring rights. This slide only ccZero. Social Media Icons adapted with permission from originals by Christopher Ross. Original images are available under GPL at http://www.thisismyurl.com/free-downloads/15-free-speech-bubble-icons-for-popular-websites
  • 3. 6/7/2014 Directorate of Soybean Research, Indore 3 Sequencing
  • 4. 1953 DNA Structure discovery 1977 2012 Sanger DNA sequencing by chain-terminating inhibitors 1984 Epstein-Barr virus (170 Kb) 1987Abi370 Sequencer 1995 2001 Homo sapiens (3.0 Gb) 2005 454 Solexa Solid 2007 2011 Ion Torrent PacBio Haemophilus influenzae (1.83 Mb) 2013 Slide credit: Aureliano Bombarely Sequencing over the Ages Illumina Illumina Hiseq X 454 6/7/2014 Directorate of Soybean Research, Indore 4 Pinus taeda (24 Gb) 2014 MinION The Next Generation
  • 5. 6/6/2014 Directorate of Soybean Research, Indore 5 Its all about the $ http://www.genome.gov/sequencingcosts/
  • 6. 6/6/2014 Directorate of Soybean Research, Indore 6 First generation sequencing
  • 7. Sanger method 6/6/2014 Directorate of Soybean Research, Indore 7 Frederick Sanger 13 Aug 1918 19 Nov 2013 Won the Nobel Prize for Chemistry in 1958 and 1980. Published the dideoxy chain termination method or Sanger method in 1977 http://dailym.ai/1f1XeTB
  • 8. Sanger method 6/6/2014 Directorate of Soybean Research, Indore 8 http://bit.ly/1g6Cudq http://bit.ly/1lcQO4J
  • 9. First generation sequencing Very high quality sequences (99.999%) Very low throughput 6/6/2014 Directorate of Soybean Research, Indore 9 Run Time Read Length Reads / Run Total nucleotides sequenced Cost / MB Capillary Sequencing (ABI3730xl) 20m-3h 400-900 bp 96 or 386 1.9-84Kb $2400 http://bit.ly/1clLps3 http://1.usa.gov/1cLqIRd
  • 10. Next generation sequencing 6/6/2014 Directorate of Soybean Research, Indore 10
  • 11. 6/6/2014 Directorate of Soybean Research, Indore 11 http://bit.ly/1keDtZQ Second generation Third generation Fourth generation Next-next-generation Next-next-next generation http://www.acgt.me/blog/2014/3/10/next-generation- sequencing-must-diepart-2
  • 12. Use the specific technology used to generate the data Illumina Hiseq/Miseq/NextSeq Pacific Biosciences RS I/RS II Ion Torrent Proton/PGM SOLiD 454 6/6/2014 Directorate of Soybean Research, Indore 12 http://www.acgt.me/blog/2014/3/10/next-generation- sequencing-must-diepart-2
  • 13. 454 Pyrosequencing One purified DNA fragment, to one bead, to one read. 6/6/2014 Directorate of Soybean Research, Indore 13 http://bit.ly/1ehwxWN GS FLX Titanium http://bit.ly/1ehAcEh
  • 14. Illumina 6/6/2014 Directorate of Soybean Research, Indore 14 Output 15 Gb 120 GB 1000 GB 1800 GB Number of Reads 25 Million 400 Million 4 Billion 6 Billion Read Length 2x300 bp 2x150 bp 2x125 bp (2x250 update mid-2014) 2x150 bp Cost $99K $250K $740K $10M Source: Illumina
  • 15. Illumina 6/6/2014 Directorate of Soybean Research, Indore 15 Output 15 Gb 120 GB 1000 GB 1800 GB Number of Reads 25 Million 400 Million 4 Billion 6 Billion Read Length 2x300 bp 2x150 bp 2x125 bp (2x250 update mid-2014) 2x150 bp Cost $99K $250K $740K $10M Source: Illumina $1000 human genome??
  • 16. Illumina 6/6/2014 Directorate of Soybean Research, Indore 16 http://1.usa.gov/1fP9ybl
  • 17. Illumina:Moleculo 6/6/2014 Directorate of Soybean Research, Indore 17 http://bit.ly/1aEPOBn
  • 18. Pacific Biosciences SMRT sequencing Single Molecule Real Time sequencing 6/6/2014 Directorate of Soybean Research, Indore 18 http://bit.ly/1naxgTe
  • 19. Pacific Biosciences SMRT sequencing Error correction methods 6/6/2014 Directorate of Soybean Research, Indore 19 Hierarchical genome-assembly process (HGAP) PBJelly Enlish et al., PLOS One. 2012 PBJelly
  • 20. 6/6/2014 Directorate of Soybean Research, Indore 20 Pacific Biosciences SMRT sequencing Read Lengths http://www.igs.umaryland.edu/labs/grc/ Mean Read Length: 8391 bp Maximum Subread Length: 24585 bp
  • 21. Oxford Nanopore 6/6/2014 Directorate of Soybean Research, Indore 21 https://www.nanoporetech.com/ No data yet Error model http://erlichya.tumblr.com/post/66376172948/hands-on- experience-with-oxford-nanopore-minion
  • 22. Others Ion Torrent Proton/PGM Nabsys SOLiD 6/6/2014 Directorate of Soybean Research, Indore 22
  • 23. Comparison 6/6/2014 Directorate of Soybean Research, Indore 23
  • 24. Next generation sequencing 6/6/2014 Directorate of Soybean Research, Indore 24 Run Time Read Length Quality Total nucleotides sequenced Cost /MB 454 Pyrosequencing 24h 700 bp Q20-Q30 0.7 GB $10 Illumina Miseq 27h 2x250bp > Q30 15 GB $0.15 Illumina Hiseq 2500 11days 2x125bp >Q30 1000 GB $0.05 Ion torrent 2h 400bp >Q20 50MB-1GB $1 Pacific Biosciences 2h 5.5-8.5kb >Q30 consensus >Q10 single 400-800MB /SMRT cell $0.33-$1 http://bit.ly/1clLps3 http://1.usa.gov/1cLqIRd
  • 25. http://omicsmaps.com/ Next Generation Genomics: World Map of High-throughput Sequencers Directorate of Soybean Research, Indore6/6/2014 25
  • 26. 6/6/2014 Directorate of Soybean Research, Indore 26 http://bit.ly/18pfUId
  • 27. Real cost of Sequencing!! Sboner, Genome Biology, 2011 6/7/2014 27Directorate of Soybean Research, Indore
  • 28. Library Types Single end Pair end (PE, 150-800 bp, Fwd:/1, Rev:/2) Mate pair (MP, 2Kb to 20 Kb) 6/6/2014 Directorate of Soybean Research, Indore 28 F F R F R 454/Roche FR Illumina Illumina Slide credit: Aureliano B

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