WG1

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• WG1
• Petri Auvinen
• DNA Sequencing and Genomics Laboratory
• Institute of Biotechnology
• University of Helsinki
• http//www.biocenter.helsinki.fi/bi/dnagen/

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Deliverables WG 1 Sequence Production Analysis

• Scientific workshops
• Design and analysis of NGS data
• Whitepaper
• Gaps between existing techniques and needs to
  analyze NGS data
• Short Term Scientific Missions
• Competitive calls, preference for early-stage
  scientists
• Community-driven wiki-webpage
• Frequently updated list of references to
  state-of-the-art designs and analysis tools
  their applications to NGS data on plants

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Working Groups
WG1 Sequence Production Analysis
Locus-sequencing variability
WG2 Sequence To Phenotype Integration
Locus-penetrance
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• Parallel Sequencing Technology
• Massive throughput
• Fast sequencing
• No cloning step
• PCR
• Currently three systems
• Genome Sequencer (http//www.454.com/,http//www.r
  oche.com)
• 454 Life Sciences, Roche
• Launched in October 2005
• Solexa (http//www.illumina.com)
• Illumina
• Launched 2006
• SOLiD (http//www.appliedbiosystems.com)
• Applied Biosystems
• Launched in October 2007

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• Genome Sequencer GS20 FLX Titanium
• 454 Life Science, Roche
• Shotgun sequencing
• No plasmid libraries
• Linkers ligated to fragments
• Emulsion PCR
• Picotiter plate, 1 600 000 wells
• Titanium 3 400 000 wells
• Pyrosequencing
• (Nyren, P. et al Anal Biochem. 1993, 208,171-5)
• Detection with sensitive CCD camera
• Read length 100 -120 bp 250 300 bp 400 bp
• Run time ca. 4,5 h 7,5 h 10h
• Raw sequence ca. 25 35 Mb/run 80 100 Mb/run
  400-600 Mb/run

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Illumina/Solexa Genome Analyzer
(http//www.illumina.com)

• Clonal Single Molecule Array technology
• Sequencing-by-synthesis technology
• Reversible terminator-based sequencing
• removable fluorescence
• Flow cell with gt 10 million clusters
• Each cluster 1,000 copies of template /cm2
• 18 samples / run
• 3 laser system (660, 635, and 532 nm)
• Read length 35 50-100 bp, 1- 2 Gb / run
• Run time 3 6 days,

Cluster Station
Flow cell
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SOLiD, Applied Biosystems(http//www.appliedbiosy
stems.com)

• Sequencing by Ligation
• emPCR
• Small beads, 1mm
• Attaching to glass slides
• Labelled probes
• Fuor colours
• 2 base encoding system
• Repeated ligation steps
• Detection with 4 Mpixel camera
• Read lenght 25-30-50 bp
• 1-2 slides / run
• 1-2 Gb / run
• Run time 5 -10 days

Shendure, J. et.al. Science 2005, 309, 1728-1732

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Next next gen technologies

• Helicos (www.helicosbio.com)
• Sequencing-by-synthesis
• No PCR amplification
• 25-90 Mb/h

• VisiGen (www.visigenbio.com)
• Real-time detection of DNA synthesis, FRET
• Intact DNA fragments
• 1Mb/sec/machine

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• Pacific Biosciences (www.pacificbiosciences.com)
• (Korlach, J. et.al. PNAS 2008, 105, 1176-81,
  Levene, MJ. et.al. Science 2003, 299, 682-86)
• Single-Molecule, Real-Time (SMRT) DNA sequencing
  technology
• SMRT chip
• Thousands zero-mode waveguides (ZMWs)
• Holes 100 nm metal film, measurement volume 20
  zeptoliters (10-21 liters)
• Real-time detection of DNA synthesis
• Fluorescent dNTPs

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Exon capture, Hodges et al 2007
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Assembly Huse S, Huber J, Morrison H, Sogin M,
Mark Welch D Accuracy and quality
of massivelyparallel DNA pyrosequencing. Genome
Biology 2007, 8R14310.1186/gb-2007-8-7-r143. Butl
er J, MacCallum I, Kleber M, Shlyakhter IA,
Belmonte MK, Lander ES, Nusbaum C, Jaffe DB
ALLPATHS De novo assembly of whole-genome
shotgun microreads. Genome Research Warren R,
Sutton G, Jones S, Holt R Assembling millions of
short DNA sequences usingSSAKE. Bioinformatics
2007, 23500-1. Zerbino D, Birney E Velvet
Algorithms for De Novo Short Read Assembly Using
DeBruijn Graphs. Genome Research 2008,
4510.1101/gr.074492.107. Simpson, J, Wong K,
Jackman S, Schein J, Jones SJM, Birol I ABySS A
parallel assembler for short read sequence data.
Genome Research (2009).doi10.1101/gr.089532.108
Annotation Parra G, Bradnam K, Korf I CEGMA a
pipeline to accurately annotate core genes in
eukaryotic genomes. Bioinformatics 2007,
10.1093/bioinformatics/btm071