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.