Illumina MiSeq Sequencing
Work flow
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
- Fragments hybridized and extended
- Denaturing of double-stranded DNA
- Bridge amplification
- Denaturing of double stranded bridge
- Bridge amplification
- Linearization
- Reverse strand cleavage
- Blocking
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
Sequencing
During sequencing, MicroGenDX runs positive and negative controls through the entire process to view any reagent contamination. Even though all our reagents and supplies are sterile, the positive controls ensure we properly identify known targets at their known concentration throughout the entire process. The negative controls ensure that we can identify any known contaminates that could have been introduced during lab procedure steps. If any contaminates are present, then they are identified and removed during the bioinformatics stage of the NGS process.
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Bioinformatic pipeline overview