Illumina MiSeq sequencing
Saskia HiltemannIllumina MiSeq sequencing is based on sequencing by synthesis. As the name suggests, fluorescent labels are measured for every base that bind at a specific moment at a specific place on a flow cell. These flow cells are covered with oligos (small single strand DNA strands). In the library preparation the DNA strands are cut into small DNA fragments (differs per kit/device) and specific pieces of DNA (adapters) are added, which are complementary to the oligos. Using bridge amplification large amounts of clusters of these DNA fragments are made. The reverse string is washed away, making the clusters single stranded. Fluorescent bases are added one by one, which emit a specific light for different bases when added. This is happening for whole clusters, so this light can be detected and this data is basecalled (translation from light to a nucleotide) to a nucleotide sequence (Read). For every base a quality score is determined and also saved per read. This process is repeated for the reverse strand on the same place on the flow cell, so the forward and reverse reads are from the same DNA strand. The forward and reversed reads are linked together and should always be processed together!
For more information watch this video from Illumina
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