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High throughput sequencing Barbera van Schaik
Bioinformatics Laboratory, AMC
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Overview Technique Applications Barbera van Schaik
Bioinformatics Laboratory
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Technique Barbera van Schaik Bioinformatics Laboratory
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Sequencing methods Synthetic chain-terminator chemistry
Sequencing by hybridisation Pyrosequencing Base-by-base sequencing by synthesis Sequencing by ligation Nanopore technology Single-molecule sequencing by synthesis in real time Synthetic chain-terminator chemistry Sanger sequencing. Old method Sequencing by hybridisation Use microarrays with different sequence combinations Pyrosequencing 454 method Sequences on beads Beads in small containers Amplification step Synthesize one type of nucleotide at the time Measure after each nucleotide. (More details in following slides) Remove label and synthesize next type of nucleotide Base-by-base sequencing by synthesis Solexa method Sequences on surface Mixture of nucleotides with different labels Make image Remove label and synthesize next base Sequencing by ligation (I do not understand the details) Ligate small pieces of nucleotide strings with different labels Nucleotides in the middle of the string correspond to the sequence Nucleotides are determined with a certain distance Make sequence shorter and repeat procedure After a few repetitions all nucleotides in a window are covered Nanopore technology Single-molecule sequencing by synthesis in real time Barbera van Schaik Bioinformatics Laboratory
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Methods Pyrosequencing (454) Pyrophosphate release after each
Bentley 2006, Curr opinion in genetics Synthetic chain-terminator chemistry Sanger sequencing Pyrosequencing (454) Pyrophosphate release after each nucleotide incorporation Sequencing by ligation (SOLiD) Nanopore technology Base-by-base sequencing by synthesis (Solexa) Sequencing by hybridisation Single-molecule sequencing by synthesis in real time Barbera van Schaik Bioinformatics Laboratory
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Illumina - Solexa Barbera van Schaik Bioinformatics Laboratory
solexa.com Barbera van Schaik Bioinformatics Laboratory
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Illumina - Solexa Barbera van Schaik Bioinformatics Laboratory
solexa.com Barbera van Schaik Bioinformatics Laboratory
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Illumina - Solexa Barbera van Schaik Bioinformatics Laboratory
solexa.com Barbera van Schaik Bioinformatics Laboratory
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Illumina - Solexa Barbera van Schaik Bioinformatics Laboratory
solexa.com Barbera van Schaik Bioinformatics Laboratory
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Roche - 454 454.com Barbera van Schaik Bioinformatics Laboratory
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Roche - 454 Barbera van Schaik Bioinformatics Laboratory
Nature Methods 2007 advertisement 454.com Barbera van Schaik Bioinformatics Laboratory
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Roche - 454 Barbera van Schaik Bioinformatics Laboratory
Pyrophosphate release after incorporation nucleotide. ATP+luciferase = light (luciferin oxidation) Nature Methods 2008 Barbera van Schaik Bioinformatics Laboratory
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Roche - 454 Barbera van Schaik Bioinformatics Laboratory
Nature Methods 2008 and 454.com Bioinformatics Laboratory
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Roche - 454 Add adaptors with different sequences
Analyze samples in one run Barbera van Schaik Bioinformatics Laboratory
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Applications Barbera van Schaik Bioinformatics Laboratory
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Overview Barbera van Schaik Bioinformatics Laboratory
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Whole genome De novo sequencing Re-sequencing Copy number variations
Rearrangements New insertions/deletions/mutations Barbera van Schaik Bioinformatics Laboratory
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Copy number variation Barbera van Schaik Bioinformatics Laboratory
The Human Genome Structural Variation Working Group, Nature 2007 Barbera van Schaik Bioinformatics Laboratory
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SNP/Haplotype analysis
Linkage studies Forensic research Barbera van Schaik Bioinformatics Laboratory
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Gene regulation studies
Chip-on-sequencing Protein-DNA Protein-RNA Barbera van Schaik Bioinformatics Laboratory
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Chip-seq Barbera van Schaik Bioinformatics Laboratory
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Gene expression Full-length transcripts EST sequencing
5' transcript ends (5'-RATE, CAGE) SAGE ditag sequencing SAGE-like 3' end sequencing Nebulized fragments ncRNA sequencing Nebulization = fragmentation with high-pressure nitrogen Barbera van Schaik Bioinformatics Laboratory
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SAGE http://www.sagenet.org Barbera van Schaik
Bioinformatics Laboratory
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CAGE Barbera van Schaik Bioinformatics Laboratory
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Epigenetics Treatment with sodium bisulfite
Unmethylated cytosines change into uracil Methylated cytosines are unchanged Compare sequences with reference sequence Barbera van Schaik Bioinformatics Laboratory
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Bisulfite sequencing Barbera van Schaik Bioinformatics Laboratory
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Sequencing of ancient DNA
Paleogenomics Sequencing of ancient DNA Mummies Sabretooth Mammoth Neanderthal Barbera van Schaik Bioinformatics Laboratory
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Metagenomics & microbial diversity
Study genomic content in a complex mixture of microorganisms (bacteria or viruses in some environment) Identify new species Barbera van Schaik Bioinformatics Laboratory
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Metagenomics Barbera van Schaik Bioinformatics Laboratory
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Sequencing in the AMC Barbera van Schaik Bioinformatics Laboratory
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Topics of interest Large-scale population studies:
mutations, cnv's, rearrangements (e.g. tumors, T- and B-cells) Other organisms (viruses, bacteria): metagenomics, whole genome (e.g. identify new bacteria, HIV1 variants) Gene expression studies: SAGE-like, multiple samples in one run Barbera van Schaik Bioinformatics Laboratory
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Barbera van Schaik Bioinformatics Laboratory
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Overview Barbera van Schaik Bioinformatics Laboratory
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