Ultrasequencing: methods and applications of the new generation sequencing platforms Nuria Tubío Santamaría Course: Genomics Universitat Autònoma de Barcelona 1
Introduction 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. 2
Sanger vs. NGS Sanger 96 sequence reads at a time In vivo cloning and amplification Read lengths: 650-800 bp High accuracy of sequencing reads NGS Millions of parallel reads at a time In vitro adaptor ligation Read lengths: 35-250 bp Lower accuracy of sequencing reads 3
Second Generation Sequencing Roche/454 FLX Pyrosequencer Illumina Genome Analyzer Applied Biosystems SOLiD Sequencer Ion Torrent Semiconductor Sequencing Helicos Sequencer 4
Roche/454 FLX Pyrosequencer 1. Emulsion PCR Image by Shendure and Ji 5
Roche/454 FLX Pyrosequencer 2. Pyrosequencing Image by Morey et al Image by Shendure and Ji 6
Illumina Genome Analyzer 1. Bridge-PCR Image by Shendure and Ji 7
Illumina Genome Analyzer 2. Sequencing by synthesis Image by Morey et al Image by Shendure and Ji 8
Applied Biosystems SOLiD Sequencer 1. Emulsion PCR 2. Sequencing by ligation Image by Morey et al 9
Applied Biosystems SOLiD Sequencer 2. Sequencing by ligation Image by Mardis et al 10
Ion Torrent Semiconductor Sequencing 1. Emulsion PCR 2. Semiconductor sequencing 11
Helicos Sequencer 1. No amplification 2. Sequencing by synthesis Image by Shedure et al 12
Compared characteristics Image by Morey et al 13
NGS applications Whole genome resequencing Targeted resequencing De novo sequencing Transcriptome sequencing Metagenomic sequencing Small RNA sequencing Methylation analysis Chromatin immunoprecipitation-sequencing (ChIP-Seq) 14
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. 15
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. 16
Single-Molecule-Real-Time sequencing Image by Morey et al 17
Nanopore Sequencing Image by Schadt et al Image by Schadt et al 18
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. 19
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