Genomics and Bioinformatics Tuesday Lecture Week 3
Genomics and Bioinformatics Tuesday Lecture Week 3 Biol 119
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This 4 page Class Notes was uploaded by Anastassia Erudaitius on Sunday April 17, 2016. The Class Notes belongs to Biol 119 at University of California Riverside taught by Dr. Hayashi and Dr. Stajich in Spring 2016. Since its upload, it has received 28 views. For similar materials see Genomics and Bioinformatics in Biology at University of California Riverside.
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Date Created: 04/17/16
Lecture 4/12/16 [Note: Dr. Hayashi at meeting] Know definition of contig and scaffold from reading o Contig – continuous stretches of DNA of sequence clones o Scaffold – Physical maps vs classical genetic maps Maps show the relationship between two genes (can include recombination frequency) Slide 4 – markers on this map are the different genes you can tell distances o If you were given this image on an exam, you could be asked how long is this genome? Answer: 19,000 bp o Distance is always measured from the origin of replication on circular maps o Humans always have linear chromosomes segment of chromosome 19 Blue represents diseases associated with that chromosome Red are [genes?] Black are [ markers?] Technology of sequencing at the time of the Human Genome Project you could maybe do 600 sequences in a set Want to make sure you get full coverage and that it is accurate can do that two ways: o Hierarchical shotgun sequencing You don’t need to know sequence information to make a map Take your “book” and sort it into pages if all you could see on a particular clone was the page you could organize the pages and then sequence a certain fragment o Whole genome shotgun sequencing (done by Celera company) In some ways the more complicated route but the less labor intensive route Take your library of genome and shred it into bits and sequence every little bit and try to assemble it from there by matching aligning sequencing (like building a puzzle back together) Need lots of copies of the genome within the library in order to have adequate coverage Must use a computer that is going to match sequences to one another, and often times yo have gaps you don’t get a complete map Contig: A A A Reads Contig Scaffold: start aligning sequences of the reads to see which portions of the sequences match, then you have a contig now if you had another set of reads you could have another contig and hopefully if you can get a third set you can assemble all of the contigs into a scaffold o The scaffold would be the joining of the 3 contigs o If every single read set is A, you can be confident that A is the nucleotide at that point – this is coverage Slide 11 – a tiling path with minimal coverage (they slightly overlap) Restriction enzyme – has a specific recognition sequence cuts the genome at specific sequences o Many of them cut at 6 bp but some can cut at 8 o These are often cut at palindromic sequences Slide 12 – human chromosome cut at different sites every 5 lane is just a marker o The challenge with this method: how do you know that a particular fragment is one fragment or two fragments there could be multiple restriction cut sites that result in the same fragment size Slide 13 – once you analyze and image them you get an assembly of how things go together o You’ve mapped those clones and then you can figure out how to sequence o If you had a sequencing project and these were your clones where would you start? Probably would want to start with the bottom black one (starting at an end) Wouldn’t want to start with the pink line because there is nothing novel from it can’t get as much information from it, so you’d probably want to skip the pink and the dark blue one Slide 14 – since the Human Genome Project they have come up with more expedient ways to do restriction enzyme mapping o The software in the machine analyzes where the florescence is and analyses almost whole chromosomes Slide 16 – you can sequence just the ends, then you might get a relationship between these contigs so you sequence the ends and then you randomly sequence that one clone o You now have sequences that show you which clones overlap with which clones o Then you take the next one and start building across o Then you take the next one that will give you coverage sequencing one that gives you more novel sequence rather than one that gives you the most coverage Hybridization assays o Rather than sequencing little bits of DNA you are taking the DNA, putting it in a tube and seeing what hybridizes to what o Similar to Northern or Southern blots o Take ssDNA and end up with florescence mapping to your clones st o 1 probe with known repetitive sequences Because you want to eliminate anything that is not unique If you were to choose something that happens to be a transposon, or retrotransposon, etc. you would be “spinning your wheels sequencing all of them” nd o 2 search for ones that didn’t line up to all these probes o 3 hybridize it to all the other clones that are unique may only overlap partially th o 4 – Then you take another set of DNA and hybridize it again to the other clones Slide 19 – Example o Randomly select a probe – probe D o Hybridize probe d to c and e o I is going to hybridize with h and j o Now we have two groups o Now select another clone that hasn’t given a positive – b o B is going to hybridize with a and c o Now you know c joins with b and d o And you can now build a bigger contig o Now pick another one – pick g – hybridize it o G hybridizes to f and h o And now you have an even larger contig o Now you should be able to tell that e and f can hybridize together o Now you probe with a and then you probe with j to see if you can continue to expand your initial contig now you have a complete contig and you can build out further from there Slide 21 – this shows what b hybridizes to and what it doesn’t hybridize to Microscope slide with probes connected to them Slide 22 – now you can organize the order of these clones The more in-depth physical map you have the more in depth genome map you can get
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