GENOME SEQUENCING. I. Genome sequencing The Sanger Method (1977) Denaturation +priming...

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GENOME SEQUENCING

Transcript of GENOME SEQUENCING. I. Genome sequencing The Sanger Method (1977) Denaturation +priming...

Page 1: GENOME SEQUENCING. I. Genome sequencing The Sanger Method (1977) Denaturation +priming Polymerization.

GENOME SEQUENCING

Page 2: GENOME SEQUENCING. I. Genome sequencing The Sanger Method (1977) Denaturation +priming Polymerization.

I. Genome sequencing

The Sanger Method (1977)

Denaturation +priming

Polymerization

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End of polymerization

I. Genome sequencing

The Sanger Method (1977)

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I. Genome sequencing

The Sanger Method (1977)

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I. Genome sequencing

The caveats of the Sanger Method

Technically cumbersome and time consuming (limited samples processing)

High amounts of starting material

Not well-suited for high-throughput analyses (genome sequencing)

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1st generation sequencing : Improvement of sequence analysis (column electrophoresis)

I. Genome sequencing

The Sanger Method (1977)

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1st generation sequencing : Improvement of sequence analysis (capillary electrophoresis)

I. Genome sequencing

The Sanger Method (1977)

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Parallel random sequencing : after fragmentation and cloning

1st generation sequencing : Improvement of the methodology

I. Genome sequencing

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2nd generation sequencing : Areas of improvement

I. Genome sequencing

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2nd generation sequencing :

Improvement of template preparation : amplification by PCR

Emulsion PCR (emPCR)

I. Genome sequencing

Used by 454 (life science), Polonator, Ion torrent and Solid

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2nd generation sequencing :

Improvement of template preparation : amplification by PCR

Bridge PCR (illumina plateform)

I. Genome sequencing

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2nd generation sequencing :

Improvement of template preparation : Single Molecule Sequencing

I. Genome sequencing

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Sequencing by reversible terminator (illumina/Solexa genome Analyzer)

2nd generation sequencing : real-time sequencing

I. Genome sequencing

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Sequencing by reversible terminator

Used in combination with bridge PCR in the illumina/Solexa genome Analyzer

2nd generation sequencing : real-time sequencing

I. Genome sequencing

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Pyrosequencing

2nd generation sequencing : real-time sequencing

I. Genome sequencing

Used in the 454 plateform (Life Sciences)

emPCR+Pyrosequencing

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Ligase-based sequencing

2nd generation sequencing : real-time sequencing

I. Genome sequencing

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Ligase-based sequencing

2nd generation sequencing : real-time sequencing

I. Genome sequencing

SOLiD DNA Sequencing Technology (Applied Biosystems)

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SMRT Sequencing Technology : Single-Molecule Real-Time Sequencing

A true continous imaging of dye-labelled nucleotides incorporation

2nd generation sequencing : real-time sequencing

I. Genome sequencing

Pacific Biosciences : SMRT : Single Molecule Real-Time Sequencing

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I. Genome sequencing

Today's technologies generate huge amount of short, high quality sequencing data

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II. Data analysis

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II. Data analysis

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III. NGS Applications

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III. NGS Applications