Hello everyone. This is the supplementary video for this week. In this week, Dr. Gao have introduced the next generation sequencing technology and algorithms involved in data analysis. Today, we come to Dr. Liping Wei's labratory in national institute of biological sciences t to bring you more perceptual knowledges about the nexgeneration sequencing. Next, we will show you the experimentation process of Ion Torrent PGM sequencer and the data generated by it. This experiment is done by Xiaoxu Yang and Sheng Wang of our lab. Ion Torrent PGM (by life technologies) came to market in 2011. It adopts Ion Torrent semiconductor sequencing technology for next- It adopts Ion Torrent semiconductor sequencing technology for next-generation sequencing, which does not depend on any optical imaging system at all. Compared to the HiSeq sequencing technology, the Ion Torrent PGM has two biggest advantages. One is the low cost. The 318 chip can sequence 1 million base pairs (or equivalently 1Mb data) with just $0.63. The other advantage is the fast workflow. It takes only 2 to 3 days to finish the whole pipeline, from library construction to the sequencing itself. This figure displays the core principles of Ion Torrent PGM sequencing. When synthesizing the nucleotide sequence, Ion Torrent will be added with four kinds of dNTPs sequentially. When a dNTP is added to the nucleotide sequence, a hydrogen ion will be released. The charge of that ion will slightly change the pH of the solution. If there are two identical bases in a row, the change of PH will be doubled. The semiconductor chip is embedded with lots of such PH sensors. It can detect the bases added to each of millions and billions of sequences simultaneously, and then determine the whole sequence. Though the core principle is simple, the whole sequencing workflow (i.e. a run) of Ion Torrent takes four steps. The first step is library preparation. Briefly, the sequence needs to be fragmented due to the machineâs limitation of read-length. Then the sequence is added to its two ends an adaptor, which is a small segment of specific DNA sequence. The second step is to prepare the template. The library fragments are bounded to the polyacrylamide beads, which are also called as the Ion Sphere particles. Then they are amplified by emulsion PCR (emPCR). It is equivalent to the clustering process of other deep sequencing technologies. The PCR will enrich a library of DNA fragments correctly flanked by the adapters. You can imagine the ISP as a small thorny ball. We coat the ball with sequences and amplify them. Then the ball will turn into an "thorny" ball full of âthornsâ, i.e. the DNA sequences. Then comes the real sequencing step. The Ion Sphere particles (ISPs) are put into a delicate semiconductor chip. The chip consists of millions of wells and each well is expected to have inside a Ion Sphere⢠particles (ISP) . By comparing the PH value of each well with the standard PH after each addition of a certain type of dNTP, we can determine which base is added, thus obtaining the whole sequence for each well. In the last step, the Ion Torrent supporting software will generate the raw data and the sequencing profiles. They are then converted to the common data formats, such as fastq, for downstream analysis. The main purpose of this supplementary material is to show how the Ion Torrent sequencing system works. It helps you to have some perceptual knowledge of modern high-throughput sequencing technologies, thus relating the abstract description in books or documents to the concrete objects. Therefore, I will not explain principles or operations that are too specific here. Here, weâll show you what the real Ion Torrent sequencer looks like, and how it works. The demonstration and experimental operation is performed mostly in the Liping Wei laboratory in the National Institute of Biological Sciences in the National Institute of Biological Sciences (NIBS), Beijing . The first step is the library preparation. The input sample for the Ion Torrent PGM system can be the purified DNA after fragmentation, the cDNA generated by reverse transcription, or mixed DNA fragments (e.g. fragments with the PCR amplicon) To construct the library for Ion Torrent PGM system, we can blunt the ends of purified DNA samples and ligate the adapters. A 5-7 cycles of enrichment will then be applied to ensure that each fragment of the library for constructing templates is flanked by two adapters simultaneously. As the video shows, this step can be done by general PCR machines in the laboratory. The next step is to prepare the template for sequencing. This step is automatically performed primarily in the Ion OneTouch 2 and Ion OneTouch ES instruments. This is the Ion OneTouch 2. This step is to amplify the library fragments on ISPs by emulsion PCR and enrich those ISP that can be used as templates. The emulsion PCR amplifies the PCR system in tiny aqueous phase droplets, which are enclosed by oil phase to avoid cross-contamination. Note that the templates are the library DNA fragments ligated with adapters; for the primer, one of its ends is bound to the ISP, and the other is in the PCR system. Now as you can see, the research staff is putting the emulsion PCR system into the Ion OneTouch instrument. The Ion OneTouch can automatically perform the generation of the emulsion, PCR amplification, and ISP centrifugal recovery. After getting the precipitated ISPs, we will use Ion OneTouch Enrichment System (ES) for automatic enrichment. This is the instrument. The instrument uses the streptavidin magnespheres for enrichment of specific sequences to recycle the ISPs that can be used as templates. After the enrichment, we add to them the sequencing primers, the polymerase, and the control ISPs, and mix thoroughly. We finally get the mixture in the tube as the video shows. Then we can add the sequencing sample into a semiconductor chip. This is the semiconductor chip. It consists of millions of wells and each chip well is expected to have inside a Ion Sphere particle (ISP). Itâs not so unimaginable to add the sequencing sample into the chip. There is a small hole on the chip. You can add slowly the sequencing sample through this hole by rotating the pipetteâs volume control knob at a speed of about 1 uL/s. Now we can see the experimenter is loading sample into the chip. After that, we can start the sequencing run. Here is the Ion Torrent PGM in our laboratory. Before sequencing, we need to initialize the instrument. As mentioned before, the sequencer identify which base is added by detecting changes in pH. So the initialization process is actually cleaning the entire sequencing system and calibrating the pH value. Then we can see the chip is being installed into the Ion Torrent PGM sequencer . We can run the sequencing after that. So we can start sequencing now. The entire sequencing process is fully automated. The four tubes in the video correspond to four kinds of free nucleotides, dATP, dTTP, dCTP and dGTP. Now we are in the process of sequencing,you can see a progress bar on the screen of the sequencer Please pay attention to the top right corner of the screen. As you can see, the sequencing process is actually to relate a type of nucleotide to wells thatsignal after each addition of a nucleotide of that type. After hundreds of the cycle, we can obtain the whole sequence of each well. The last part is the data generation. Once the raw signal data is generated on the Ion PGM sequencer, it is automatically transferred to the connected Torrent Server. Using the Ion Suite developed by Ion Torrent, we can login into the Torrent Server via a browser to check all the summaries and history records. We can also check the quality of each sequencing, such as the percentage of ISPs that are loaded, the percentage of enriched templates, etc. As you can see from the video, each raw signal strength files output by Ion Torrent corresponds to the signal change at each point when each base flows in(which means that the number of flows corresponds to the number of dat files, and the files are very big). However the preliminary internal data processing will transform dat files into SSFs (Standard Flowgram Files) as the first operable data. Let us to see a sequence record There are the summary of sample loading , the quality of reads etc. we won't go into it further there Letâs get back to this screen. The heat map on the screen corresponds to the loading status of a chip. The red color means that an ISP is loaded, while the blue means not. Here we can see there's a small bubble in the middle of the chip. Next is a summary of the loading status, including the percentage of ISPs that are loaded, the percentage of template-positive ISPs, etc.. The number of reads differs between sequencing chips with different throughputs.. The total number of reads equals more or less to the number of wells in a chip * the percentage of ISPs that are loaded * the percentage of template-positive ISPs * (1 â the percentage of control ISPs). The length of reads varies between 100bp and 450bp as different sequencing kits are used. Because this is not a tutorial, details of data assessment will not be explained here. Students who are interested can post and discuss in the forum. OK, thatâs all for the introduction of Ion Torrent. We hope itâs helpful for you. Thanks!
