Next Generation Sequencing

Next Generation Sequencing

contact: ludwig@univ-brest.fr

1. Variants

1.1. What is a Variant ?

1.1.1. Small sized Variations

  • SNV: Single Nucleotide Variant (abusively simplified as SNP: Single Nucleotide Polymorphisme) AGTGTGC → AGTATGC
  • Short indel: insertion or deleltion
    • Insertion: AGTGTGC → AGTGACGCTGC
    • Deletion: AGTGTGC → AGTC

1.1.2. Structural Variantions

1.2. Why look at variants ?

  • Identification (Genotyping, not Sequencing)
    • Filiation
    • Investigation
    • Archeology
  • Medicine
    • Clinical diagnostics
    • Cancerology
    • Personalized Medicine
  • Research
    • Genetic Diseases
    • Population Genetics
    • Ancient DNA Studies

2. Sequencing Steps: from Biological Fragments to Numerical Reads

2.1. Library Preparation

  1. Amplification: Making copies of the DNA to sequence
    amplification.png
  2. Fragmentation: Division in double stranded Fragments of different sizes
    • Sonication
    • Nebulization
    • Enzyme Digestion

fragmentation.png

  1. Ligation: Extension of the Fragments with Double Stranded DNA Adapters (Oligonucleotides)
    • Adapters allow for Hybridization to the flow Cell

ligation.png

  1. Denaturation: to get 2 Single-Stranded Fragments from 1 Double-Stranded Fragment

denaturation.png

2.2. Amplification (multiple identical copy)

  1. Binding to the Chip (Hybridization to the Flow Cell) Either on 5' or '3' (pairing to the Adapters in the Flow Cell)
    hybridization.png

  1. DNA Amplification by PCR: Production of the complementary strand from the Flow Cell Adapters
    polymerase.png

  2. Denaturation: Strand not attached to the Flow Cell is washed away
    hybriddenat.png

  1. Bridge Amplification: great number of cycles of PCR + Denaturation
    bridgeall.png

  2. Keeping only Single-Stranded Fragments corresponding to the forward strands
    washing.png

2.3. Sequencing By Synthesis

  1. Forward Strand is sequenced by synthesizing a complementary strand with marked nucleotides
  2. A light source excites the fluorescent marker and the emitted wave length is read
  3. This light emission is then converted to a base call
  4. A Bridge is formed again and this time, after denaturation, the forward Strand is washed away and the reverse strand is sequenced
  5. Image files are converted to text files (FastQ)
Conclusion

Sequencing takes biological Fragments as input and produces numerical Reads (or Read Pairs)

paired-end-read.jpg

3. Workflow: From Reads to Variants

gatkBestPractices.png