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NGS Data Generation

NGS Data Generation. Dr Laura Emery. Overview. The NGS data explosion Sequencing technologies An example of a sequencing workflow Bioinformatics challenges. The NGS data explosion. EBI biological data. TB of data. Bottlenecks to biological research. Source: Qiagen. NGS Technologies.

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NGS Data Generation

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  1. NGS Data Generation Dr Laura Emery

  2. Overview • The NGS data explosion • Sequencing technologies • An example of a sequencing workflow • Bioinformatics challenges

  3. The NGS data explosion

  4. EBI biological data TB of data

  5. Bottlenecks to biological research Source: Qiagen

  6. NGS Technologies • A variety of platforms available • Differ in: • Library preparation • Sequencing chemistry

  7. Comparison of NGS Technologies

  8. Example: Illumina NGS workflow

  9. 1. Library preparation • RNA extraction RNA only • Fragmentation and size selection • cDNAsynthesis RNA only • Adapter ligation

  10. 1. Library preparation • Alternative library preparation methods: • Mate pair • Targeted • Strand specific

  11. 1. Library preparation • Multiplexing (optional)

  12. Example: Illumina NGS workflow

  13. 2. Hybridisation and Amplification

  14. Example: Illumina NGS workflow

  15. 3. Sequencing Errors!

  16. 3. Sequencing (Paired-end)

  17. Example: Illumina NGS workflow

  18. Other • data 4. Data analyses: generalised pipeline

  19. Bioinformatics challenges • Library preparation biases • Random hexamer priming • GC content • Data storage • Data analysis • Errors • Mapping/assembly uncertainty

  20. Bioinformatics challenges • Library preparation biases • Random hexamer priming • GC content • Data storage • Data analysis • Errors • Mapping/assembly uncertainty Sequence bias in the first 13 nucleotides Methods for correction: Cufflinks, mmseq

  21. Bioinformatics challenges • Library preparation biases • Random hexamer priming • GC content • Data storage • Data analysis • Errors • Mapping/assembly uncertainty GC-rich or AT-rich fragments have been found to be over/underrepresented Methods for correction: EDASeq, CG correct

  22. Bioinformatics challenges • Library preparation biases • Random hexamer priming • GC content • Data storage • Data analysis • Errors • Mapping/assembly uncertainty

  23. Conclusions • NGS technologies provide us with new opportunities but new challenges • You will learn more about overcoming these challenges during this course • Furthermore, other omics technologies will be introduced

  24. So over to Bernardo…

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