Hello, everyone!
The supplementary video mainly introduces the Hiseq and Miseq high-throughput sequencing platform developed by Illumia company.
Although it's about two platforms: Hiseq and Miseq,
but in terms of sequencing principle , they are the same platform.
They adopt the consistent workflows and sequencing techniques.
To some degree, Miseq is an integrated compressed version of Hiseq
The slices in the video are mainly some training materials provided by Illumina company. The demonstration experiments are primarily
completed in National Institute of Biology Sciences, Beijing and School of Life Sciences, Peking University.
First, we¬タルll introduce the basic workflow and sequencing principles of the two platforms.
HiSeq and Miseq sequencing system adopt the Sequencing-By-Synthesis (SBS) technology
and a new sequencing technology using reversible terminator chemistry.
Before sequencing,
the sequencing library is attached to the optically transparent small chip, also known as Flowcell.
After the bridge amplification, the attached library fragment forms hundreds of millions of clusters on the flowcell.
Each cluster is a single molecule cluster of thousands of copies.
By using the four types of dNTPs with fluorophore, which can combine with the reversible terminator,
it can realize the synthesis of only one base at a time by the SBS technology of reversible termination.
Then it uses the corresponding laser to excite the fluorophore and captures the excitation light.
Thereby it can read the base information and sequence the DNA template.
Here we'll introduce the whole workflow step by step.
The first is the library preparation.
Samples for sequencing can be the interrupted genomic DNA, cDNA generated by reverse transcription or PCR amplified products, etc.
After joining each of the sequence ends with an adaptor, we complete the library preparation.
The adaptor contains the sequence complementary with the oligo array on the flowcell.
The figure shows a typical structure of the fragment to be sequenced. Its both ends comprise the primer P5 and P7,
which connects to the flow cell, indics and the sequencing primers.
The prepared library needs to pass the quality detection to be applied to sequencing.
Quality detection generally includes detecting library fragment size distribution,
and using real-time PCR kit to detect the molar concentration of the structurally normal library fragment.
The next step is the cluster generation.
Hiseq has a dedicated cluster generation system cBot for sequencing library clonal amplification. Instead,
Miseq integrates the cluster generation system and the sequencing system on the same machine.
The cluster generation is actually a DNA fragment enrichment process. It mainly ligates a DNA fragment in the library vertically on a flow cell. Then it amplifies the fragment by the bridge PCR to enrich to clusters.
The flow cell is a chip. As the figure shows, the DNA fragment in the library is firstly ligated vertically on a flow cell.
After the bridge PCR, each DNA fragment will be amplified many times in the cBot, forming clusters of the DNA fragment, which are actually copies of the DNA fragment.
In the Hiseq platform, this process is mainly completed in the cBot. And this is cBot.
The operation of cBot is relatively simple. The first step is to use MiliQ liquid water to clean the cBot¬タルs liquid road system.
Now we can see that our research staffs are cleaning the fluid path system.
This is the flow cell, and we¬タルll install it on the cBot machine.