A big welcome to “Bioinformatics: Introduction and Methods” from Peking University! In this MOOC you will become familiar with the concepts and computational methods in the exciting interdisciplinary field of bioinformatics and their applications in biology, the knowledge and skills in bioinformatics you acquired will help you in your future study and research. Course materials are available under the CC BY-NC-SA License.
A quick tour to sequencer - illumina HiSeq & MiSeq (English Subtitles)
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Bioinformatics: Introduction and Methods 生物信息学: 导论与方法
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Prediction and Analysis of Noncoding RNA
Upon completion of this module, you will be able to: Analyze non-coding RNAs from transcriptome data; identify long noncoding RNA (lncRNA) from NGS data and predict their functions.
Conheça os instrutores
Ge Gao 高歌, Ph.D.Assistant Professor, Principle Investigator
Center for Bioinformatics, School of Life Science
Liping Wei 魏丽萍, Ph.D.Professor, Director
Center for Bioinformatics, School of Life Sciences
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.
Then, following the prompts of the instrument, we install the prepared reagent rack.
Because the process of enrichment is the PCR, the reagent rack is actually
some buffer and enzymes for hybridization, elution and amplification.
In the clustering process,
the machine will automatically follow certain procedures to continually flow the reagent through the flow cell to react.
After connecting the fluid path system, we put the sample in the sample cell
.Press the start button on the touch screen, and the clustering process will be automatically on.
The clustering process will be automated.
The clustering process will be completed after about four hours.
After completing the clustering process, we can take out the flow cell.
This moment the flow cell has been fully covered with DNA clusters. Each cluster is composed of thousands of copies of a Single-molecule copy.
Then we can apply the Hiseq 2500 for sequencing.
This is Hiseq 2500.
Now we just need to put the appropriate sequencing reagents into the reagent tank.
These sequencing reagents are also supporting and do not require the user's own preparation.
Then we transfer the flowcell processed by cBot to the flowcell slot of Hiseq sequencing instrument.
Finally we follow the prompts on the screen and confirm some of the sequencing strategies.
Then we can click the Start Button to begin sequencing.
Now we can see that Hiseq has started its run. It is also a fully automated process.
In addition to regularly monitoring the output sequencing data on the screen, it does not require additional operations.
Now we￢ﾀﾙll introduce the Miseq.
Miseq is in fact an integrated compressed version of Hiseq.
Its clustering process and sequencing process is completed on the same machine. And this is the Miseq sequencing instrument.
Miseq simplifies the whole operation process. There is only one matching kit,
as shown in the video. When the kit and samples are ready, they can be installed directly on the MiSeq.
This is the flow cell used in Miseq. What's different with Hiseq is that
the flow cell used in Miseq only has one lane while the one used in Hiseq has eight lanes.
Simply, Miseq can only sequence one library at a time. It's one of the reasons for its low throughput.
Preprocess the flow cell and install it to the flow cell slots of Miseq.
After checking that all the preparatory steps are completed, you can click the start button to start sequencing.
After entering the sequencing process, we can only monitor the status and progress of sequencing through the screen.
For example, the video shows some of the summary of this run. From it you can get the current amount of data generated, the error rate,
You can also see the density of clusters on the flow cell, the current ratio of four bases and some else in the form of charts.
And we'll not illustrate it in detail here.
The last part is the data generation.
Hiseq and Miseq sequencing instrument collect data both based on the optical system photographing the emitted fluorescence in the
based on the optical system photographing the emitted fluorescence in the sequencing process.
Therefore, the most original file is an image file with the extension of Tif.
The file stores the image information of the fluorescence when thelaser irradiates the clusters on the flowcell in each sequencing cycle.
Tif files are huge and it is impossible to save them all. So we need to convert the tif files to cif and clocs files after image analysis.
Cif files store the information of cluster fluorescence intensity. And clocs files store the cluster location information.
Then, these information will produce bcl files by the process of base calling.
This procedure is done by cluster fluorescence analysis and mathematical modeling, converting fluorescence signal to base information.
Bcl files will further be converted to fastq files. They store the read's basic information,
sequence information and the base quality of each base in the sequence.
Typically, the above process is completed by Hiseq or Miseq￢ﾀﾙs supporting software.
If you are a normal user, you'll normally contact fastq files for bioinformatics subsequent analysis.
Well, that is all for the introduction. Thanks!
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