ULTRASEQUENCING.
Next Generation Sequencing: methods and applications.
Genòmica i Proteòmica
Curs 12/13
Pablo Lammers
NIU 1323099
Sanger sequencing
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Since 1975. Frederick Sanger
Human Genome Project
New necessities
 1 sample -> 1 read -> 3 to 9€
 1 read -> 1 kbp (max.)
 1 run -> 16/48/96 samples
Next Generation Sequencing
Libraries preparation
Amplification
DNA
Sonication
Fragmentation
Physical methods
Chemical methods
Adapters ligation
Sequencing reaction
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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
Applied Biosystems: SOLiD
Illumina - MiSeq
Invitrogen – Ion Torrent
Pacific Biosciences – PacBio RS II
454 (Roche)
1.
One fragment = One bead
2.
emPCR: Emulsion PCR amplification
3.
Sequencing: One bead = One read
4.
Pyrosequencing
 1 M reads/run
 Read lenght: 250-500 bp
Library construction
DNA captured bead
containing millions of
copies of a single clonally
amplified fragment
emPCR
PTP loading
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
Sequencing by synthesis
1. Library preparation
2. Clusters generation
3. Sequencing
DNA fragmentation
Adapter oligos ligated
Purification
Isothermal bridge amplification
 100 M reads/run
 Read lenght: 80-250 bp
Ion Torrent
wells -> chemical info from DNA seq -> into digital info (basecalls)
DNA fragmented
Attached to beads
Each bead in a well
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
 Read lenght: 4 kbp
 Maximum: 23 kbp
Each nucleotide labeled with a different colored fluorosphore
Phospholinked nucleotides ->
introduced into the ZMW chamber
Base held -> light pulse produced
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.