1. ULTRASEQUENCING. Next Generation Sequencing: methods and applications.
Genòmica i Proteòmica Pablo Lammers
Curs 12/ NIU

2. Sanger sequencing 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

3. Next Generation Sequencing
Libraries preparation
DNA
Sonication
Physical methods
Fragmentation
Amplification
Chemical methods
Adapters ligation
Sequencing reaction
1 run -> 1000 €
1 read -> 100 to 400 bp
1 run -> >100 M reads
1 run -> 24 small genomes

4. NGS platforms 454 Roche – GS Junior Applied Biosystems: SOLiD
Illumina - MiSeq
Invitrogen – Ion Torrent
Pacific Biosciences – PacBio RS II

5. 454 (Roche) One fragment = One bead emPCR: Emulsion PCR amplification
Sequencing: One bead = One read
Pyrosequencing
1 M reads/run
Read lenght: bp
DNA captured bead containing millions of copies of a single clonally amplified fragment
Library construction
emPCR
PTP loading

6. Applied Biosystems: SOLiD
Amplification by emPCR
Hybridization to beads. Beads covalently attached to glass slide.
Ligation Based Sequencing with Di-Base probes (fluorescently labeled with 4 dyes)
Image capture (fluorophore)
M reads/run
Read lenght: bp

7. Illumina 1. Library preparation 2. Clusters generation 3. Sequencing
Sequencing by synthesis
1. Library preparation
2. Clusters generation
3. Sequencing
DNA fragmentation
Adapter oligos ligated
Isothermal bridge amplification
Purification
100 M reads/run
Read lenght: bp

8. Ion Torrent Nucleotide incorporated to a single DNA strain
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: bp

9. 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
Each nucleotide labeled with a different colored fluorosphore
Read lenght: 4 kbp
Maximum: 23 kbp
Phospholinked nucleotides -> introduced into the ZMW chamber
Base held -> light pulse produced

10. Applications Cancer research Population genomics studies Metagenomics
RNA-seq
Comparative genomics
Disease association studies
Species clasification
Forensics