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ULTRASEQUENCING . Next Generation Sequencing : methods and applications . Genòmica i Proteòmica Pablo Lammers - PowerPoint PPT Presentation
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ULTRASEQUENCING.Next Generation Sequencing: methods and applications.
Genòmica i Proteòmica Pablo LammersCurs 12/13 NIU 1323099
- Since 1975. Frederick Sanger- Human Genome Project- New necessities
Sanger sequencing
1 sample -> 1 read -> 3 to 9€ 1 read -> 1 kbp (max.) 1 run -> 16/48/96 samples
Next Generation Sequencing
Libraries preparation
Amplification
Sequencing reaction
DNA
Fragmentation
Adapters ligation
Sonication
Physical methods
Chemical methods
1 run -> 1000 € 1 read -> 100 to 400 bp 1 run -> >100 M reads 1 run -> 24 small genomes
NGS platforms
454 Roche – GS Junior
Illumina - MiSeq
Pacific Biosciences – PacBio RS IIInvitrogen – Ion Torrent
Applied Biosystems: SOLiD
454 (Roche)
1. One fragment = One bead
2. emPCR: Emulsion PCR amplification
3. Sequencing: One bead = One read
4. Pyrosequencing
DNA captured bead containing millions of
copies of a single clonally amplified fragment
emPCR PTP loadingLibrary construction
1 M reads/run Read lenght: 250-500 bp
Applied Biosystems: SOLiD
1. Amplification by emPCR
2. Hybridization to beads. Beads covalently attached
to glass slide.
3. Ligation Based Sequencing with Di-Base probes
(fluorescently labeled with 4 dyes)
4. Image capture (fluorophore)
100-500 M reads/run Read lenght: 50-100 bp
Illumina
2. Clusters generation1. Library preparation 3. Sequencing
100 M reads/run Read lenght: 80-250 bp
DNA fragmentation
Adapter oligos ligated
PurificationIsothermal bridge amplification
Sequencing by synthesis
Ion Torrent
DNA fragmented Attached to beads Each bead in a well
wells -> chemical info from DNA seq -> into digital info (basecalls)
one of the 4 nucleotides
Nucleotide incorporated to a single DNA strain
ion H released
pH chemichal changes -> into voltage
• each 15 sec -> wash and repeat (different nucleotide)
10 M – 1 G reads/run Read lenght: 200-400 bp
Pacific Biosystems (PACBIO)
• Amplification not required• SMRT: Single Molecule Real Time seq• ZMW: Zero-mode waveguide
DNA template-polymerase complex -> immobilized at the bottom of the ZMW
Phospholinked nucleotides -> introduced into the ZMW chamber
Each nucleotide labeled with a different colored fluorosphore
Base held -> light pulse produced Read lenght: 4 kbp Maximum: 23 kbp
Applications
•Cancer research
•Population genomics studies
•Metagenomics
•RNA-seq
•Comparative genomics
•Disease association studies
•Species clasification
•Forensics
Bibliography
• Michael A Quail et al. 2012. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 2012; 13:341. Review.
• Mardis ER. 2008. Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402. Review.
• Schuster 2008. Next-generation sequencing transforms today’s biology. Nature Methods - 5, 16 - 18 (2008). Published online: 19 December 2007; | doi:10.1038/nmeth1156.