MICROBIOLOGY | Illumina MiSeq NGS

Work flow


library preparation

Library
preparation

  • Adaptors added to DNA fragments
  • Amplification of the universal genes 16S and ITS
  • Sequencing of binding sites, indexes, and regions complimentary to oligos
  • Each plate has three positive amplification controls that span the analytic range, and a negative control for each assay
  • Specimens are combined based on qualitative measures
  • Fragment size selection and clean-up is performed to remove primer dimers, salts, and short fragments

cluster generation

Cluster
generation

  • Fragments hybridized and extended
  • Denaturing of double-stranded DNA
  • Bridge amplification
  • Denaturing of double stranded bridge
  • Bridge amplification
  • Linearization
  • Reverse strand cleavage
  • Blocking

sequencing

Sequencing

  • Following cluster generation, the sequencing primer hybridizes to the adapter sequence
  • A single fluorescently labeled deoxynucleotide triphosphate, dNTP, is added to each strand
  • Each nucleotide base has a different fluorescent dye, which allows them to be added to the reaction simultaneously, and an image is captured at each cycle
  • A terminator at the end of each nucleotide must be enzymatically cleaved before another nucleotide can be incorporated, which resolves the problem seen with homopolymers and repetitive sequences
  • Once the forward strand has been read, a bridge PCR is performed and sequencing along the reverse stand begins

Translate »
Shopping Cart
There are no products in the cart!
Continue Shopping
0