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NGS presentations – summary1. Illumina Sequencing
1.1 Steps
1) Library preparation
Tagmentation:
- DNA extraction + purification
- DNA fragmentation
- Adapter ligation (tag)
2) Cluster generation Figure 1: Tagmentation
- Capture of single stranded library fragments
- Bridge amplification millions of clonal clusters formed
3) Sequencing by synthesis
- Fluorescently labelled deoxynucleotide triphosphates (dNTP’s) added
- Each cycle: identification of nucleotide by emission wavelength
4) Data analysis
- Comparing reads to reference genome
Figure 3: Principle Illumina Figure 2: sequencing by synthesis
Figure 4: Principle Illumina
1
, 1.2 Long read principle
Figure 5: Long read principle
1.3 Advantages
- High throughput
- High speed
- Cost effective (low cast per base, multiplexing)
- Accurate base calling
1.4 Disadvantages
- Limited by size of fragments
- No real-time data access
- Signal decay and dephasing due to incomplete fluorescent label cleaving
- Lagging strand dephasing poor sequence quality towards the end
- Expensive equipment
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