ultrasequencing: methods and applications of the new...

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Ultrasequencing: methods and applications of the new generation sequencing platforms

Nuria Tubío Santamaría

Course: Genomics

Universitat Autònoma de Barcelona

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IntroductionIntroduction

Clasical methods of sequencing: Sanger sequencing.

Low throughput, large studies can´t be performed with this methodology.

In the last few years the Next Generation Sequencing (NGS) technologies have been developed.

Sequence a whole genome with a declining cost of time and money.

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Sanger vs. NGS

96 sequence reads at a time

In vivo cloning and amplification

Read lengths: 650-800 bp

High accuracy of sequencing reads

Millions of parallel reads at a time

In vitro adaptor ligation

Read lengths: 35-250 bp

Lower accuracy of sequencing reads

Sanger NGS

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Second Generation Sequencing

Roche/454 FLX Pyrosequencer

Illumina Genome Analyzer

Applied Biosystems SOLiD Sequencer

Ion Torrent Semiconductor Sequencing

Helicos Sequencer

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Roche/454 FLX Pyrosequencer

1. Emulsion PCR

Image by Shendure and Ji

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Roche/454 FLX Pyrosequencer

2. Pyrosequencing

Image by Shendure and Ji

Image by Morey et al

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Illumina Genome Analyzer

1. Bridge-PCR

Image by Shendure and Ji

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Illumina Genome Analyzer

2. Sequencing by synthesisImage by Morey et al

Image by Shendure and Ji

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Applied Biosystems SOLiD Sequencer

1. Emulsion PCR

2. Sequencing by ligation

Image by Morey et al

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2. Sequencing by ligation

Applied Biosystems SOLiD Sequencer

Image by Mardis et al

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Ion Torrent Semiconductor Sequencing

1. Emulsion PCR

2. Semiconductor sequencing

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Helicos Sequencer

1. No amplification

2. Sequencing by synthesis

Image by Shedure et al

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Compared characteristics

Image by Morey et al

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NGS applications

Whole genome resequencing

Targeted resequencing

De novo sequencing

Transcriptome sequencing

Metagenomic sequencing

Small RNA sequencing

Methylation analysis

Chromatin immunoprecipitation-sequencing (ChIP-Seq)

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Second Generation Sequencing limitations

Short read lengths due to noise and sequencing errors as the read extends (dephasing).

PCR amplifications can lead to errors in the template sequence and increase time and complexity of sample preparation.

High throughput combinated with short reads generates mountains of data that challenge storage and informatic operations.

Short reads also make alignment and assembly processes challenging.

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Third generation sequencing

Ability to sequence single molecules of DNA without need of amplification.

Detection techniques are based mainly on the physical recognition of DNA bases, rather than on detection of chemical incorporation.

Sequencing reaction is not paused for ‘wash and scan’ steps after the incorporation of each base.

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Single-Molecule-Real-Time sequencing

Image by Morey et al

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Nanopore Sequencing

Image by Schadt et al

Image by Schadt et al

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Conclusions

SGS have supposed a revolution in life sciences, allowing us to understand the complexity of the living systems.

TGS are emerging and still have to demostrate that all of the underlying sophisticated machinery in which are based can be translated into useful data.

TGS may bring ever higher dimensional data so that we can have more complete understanding of living systems and the complex phenotypes (like human disease) that emerge from such systems.

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Bibliography

.Morey M, Fernández-Marmiesse A, Castiñeiras D, Fraga JM, Couce ML, Cocho JA. 2013. A glimpse into past, present, and future DNA sequencing. Mol Genet Metab. 110(1-2):3-24.

Schadt EE, Turner S, Kasarskis A. 2010. A window into third-generation sequencing. Hum Mol Genet. 19(R2):R227-40. Erratum in: Hum Mol Genet. 2011 Feb 15;20(4):853.

Mardis ER. 2008. Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402. Review.

Mardis ER. 2008. The impact of next-generation sequencing technology on genetics. Trends Genet. 24(3):133-41. Review.

Shendure and Ji. 2008 Next-generation DNA sequencing. Nat Biotechnol. 26(10):1135-45.