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Stanford CS 262 - Lecture Notes

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Page 1 of 20 Scribed by: Joni Fazo CS262:Lecture 12 – Molecular Evolution and Phylogenic Trees I. Some New Sequencing Technologies In this lecture, we will learn about several new sequencing technologies that are currently under development, or have been made available in the last couple of years. A number of these technologies are useful for resequencing. Resequencing is the process of sequencing DNA in order to compare to a reference sequence. The goal of this process is to better understand population diversity within a species. Rather then looking for long reads that are useful for doing assembly, the focus of resequencing is on getting just enough information to anchor a given subsequence within a reference sequence in order to study the differences between the subsequence (generated by the resequencing technology) and the pre-existing refererence sequence. A. Molecular Inversion Probes One very efficient and convenient technology developed within the last few years, and made commercial last year by Affymetrix, is molecular inversion probes. This technology was actually developed by the company ParAllele BioScience, who was later aquired by Affymetrix. Molecular inversion probe technology involves constructing one probe in a circular DNA piece that contains a gap. The probe is complementary to the reference sequence with one nucleotide letter missing. Given the original genome, and the probe, the probe attaches to the right place with the one gap. A given nucleotide (A, C, T or G) is then added to the reaction, if the added nucleotide is the right one, it will close the gap, the probe will be release and then amplified, and finally detected.Page 2 of 20 This process may be parallelized in a very high throughput fashion. Hundreds of thousands of these probes may be generated for different locations in the DNA. These probes are then passed through containers labeled by A, C, T or G. Next the probes are hybridized to RNA, each location of which contains a different sequence, so the right probe goes to that location. In the last step, which of the four letters was incorporated is detected. In summary, what we can do is for a very large number of locations in the DNA we can detect whether that location contains an A, C, T or G. This technology is used by pre-constructing chips with 500,000 (or one million in the newer chips) specific locations in the human genome that can be probed this way. Those are the locations in the human genome where we know there is a lot of variation between individuals. Note - Each one of us has roughly three million differences in our genomes that are correlated at nearby locations. So by probing about one million locations of the Human Genome we can create enough of a signature to know roughly what a person’s whole genome looks like. This technology is in use and for a few hundred dollars you may get your whole genome genotyped. The technology is not perfect – many things are not detected by this technology. For example, base pair differences, gaps and inversions in other locations will not be detected. However, this is a big step towards getting personalized genomics. See http://www.affymetrix.com/technology/mip_technology.affx for more information. B. Single Molecule Array for Genotyping - Solexa Another technology is the Solexa platform that is use experimentally in a few labs such as the Broad Institute.Page 3 of 20 The idea is to shear DNA, to cut it into many short pieces and then prepare them with an adapter on each end. The pieces are then attached to a glass slide, with each end stuck to the glass to form a bridge. Primers are then used to amplify the DNA fragments. Then one end of each fragment is released resulting in single-stranded DNA stuck to the glass in clumps. Labeled nucleotides are added. A camera then reads off the results. Depending on the color of the nucleotide, the camera is able to figure out the letter of that fragment. Solexa uses this technology to obtain DNA reads from 25 – 50 nucleotides long. This is useful because it is extremely cheap and has high-throughput. Molecular inversion probes produce one letter of DNA at a specific location. With Solexa technology, we get 25 to 50 letters that can then be aligned to the reference genome (for example the Human Genome – which the technology will be used extensively on) and then figure out a single base difference not just in one location, but in arbitrary place difference or gaps, etc. The benefit is to make human resequencing possible at arbitrary locations for less then $1000. See http://www.illumina.com/pages.ilmn?ID=203 for a demo of this technology. C. Nanopore Sequencing Another technology for getting very long reads utilizes a technique of passing long single-stranded fragments of DNA through a nanopore that detects each nucleotide as it passes. Depending on which nucleotide passes the sensor detects which nucleotide has passed. This technology was hyped a couple of years ago, but is not currently considered one of the main players in new sequencing technologies. Time will tell. For more information see: http://www.mcb.harvard.edu/branton/index.htmPage 4 of 20 D. Pyrosequencing on a Chip Pyrosequencing is another technology that is currently being used extensively. This is a different reaction then gel electrophoresis. The main idea is run a series of reactions that produce light through the incorporation of one nucleotide that is detected by a camera.Page 5 of 20 This technology is applied on a massive scale by taking lots of tiny fragments of DNA, attached to magnetic beads. The beads are then distributed in a chip that contains wells, with typically 200,000 to 400,000 wells on one chip. Then nucleotides are added one after another through a process of flowing nucleotides, and then washing, flowing nucleotides and then washing. In each step only one type of nucleotide is added, then a a camera takes a picture detecting light in each well where the given nucleotide was incorporated. These reactions go for typically 200 cycles without a problem, so reads that come out of this technology (from for example the 454 platform which costs approximately $500K) can be 200 long. The next generation 454 machine is supposed to produce 16 megabases of DNA in 200 long reads for approximately $500. This technology produces lots of data with long reads that are useful for the assembly of bacteria or yeast.


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Stanford CS 262 - Lecture Notes

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