Next Generation Sequencing

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4 Terms

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What to Sequence and Why?

  • De novo whole genome sequencing: requires de novo whole genome assembly

  • Polymorphism discovery (distinct from genotyping!): Targeted approaches, Whole genome, SNPs, copy number variations, insertions, deletions, etc.

  • Expressed sequence discovery: ESTs, cDNAs, miRNAs, etc

  • Functional genomics: ChIP, Expression profiling, Nucleosome positioning

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Starting material for NGS

  • DNA (DNA-seq)

  • RNA (RNA-seq)

  • DNA fragments bound to selected protein – to analyse the sequences of DNA-binding sites of protein of interest or localisation of histone modifications (ChIP-seq)

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NGS workflow

  1. library preparation

  2. sequencing principles by synthesis

    1. Sanger/Dideoxy chain termination (Life Technologies, Applied Biosystems),

    2. Pyrosequencing (Roche/454)

    3. Reversible terminator

    4. Ion torrent

  3. sequencing by oligoligation detection like direct reading of dna sequence

    1. nanopore

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3rd generation ngs

  • single molecule real time

  • minion / gridion