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.