Genomic library prep

This past week I got started building genomic libraries for several projects that are just getting underway. It’s been interesting getting back to wetlab work, as I’ve been focusing more on several computational research projects since starting my postdoc. Two of the projects I am collecting data for are investigations of hybridization and species limits in whiptail lizards from Mexico. These are really interesting lizards that are widely distributed across North America, and are probably best known for the fact that they frequently hybridize, and in doing so sometimes produce new unisexual hybrid species! These are collaborative efforts with Adrián Nieto-Montes de Oca from UNAM who is on sabbatical in the lab this semester. Our goal is to try and clarify the complex population genetic processes that have shaped their genomes so that we can better understand their evolutionary history.

 

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Aspidoscelis guttata (a species from Southern Mexico)

The other project is on map turtles of the genus Graptemys in the southeastern U.S.. Bob worked on these guys for his dissertation, and we are collecting data to try and identify how population demographic history has shaped genetic diversity in this group. Specifically, we are trying to understand whether some of the genetic patterns we see result from hybridization between species, or instead from how more basic population genetic processes (a.k.a. lineage sorting). The ultimate goal is to sequence several thousand genetic loci for a large number of individuals of several different species within these groups. The basic steps for doing this are: 1) extract the DNA from tissue samples of the individuals you want to use in the project, 2) cut up these full genomic DNA samples using a restriction enzyme, 3) attach a unique DNA barcode to each sequence so that you can identify which individual they belong to, 4) Select a subset of the DNA fragments based on size (since we don’t need to sequence the whole genome, just a fraction of it for all our samples), 5) Amplify the fragments using pcr, 6) clean up the solution by removing other reagents so that the sample just consists of the DNA fragments in solution, and 7) sequence all the fragments using a cool, new genetic sequencing technology.

The data you get back from the sequencing facility consists of hundred of millions of DNA sequence reads that you need to assemble in order to reconstruct the genetic sequence for each individual at each genetic locus in the genome. As you can imagine, this requires quite a bit of computational power and some programming skills in order to efficiently automate the process. Adrián is here using his sabbatical as an opportunity to hone his bioinformatic skills for processing and analyzing these types of datasets, so we’ve been working with some other RADseq datasets that he brought with him as well. These include one phylogenetics project aimed at resolving the evolutionary relationships among knob-scaled lizards, and another on Anoles in southern Mexico. One thing I don’t miss about lab work is the magnetic bead cleanups in which we isolate the DNA in our genetic library. They are pretty neat the first time you do them, check out this YouTube video that show’s how quickly the beads bind to a magnet so that you can isolate the DNA and put it into a new solution. However, they can be rather frustrating when you have to do 16 at a time.

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This is what 16 pools of DNA samples bound by magnetic beads looks like!

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One thought on “Genomic library prep

  1. Pingback: Summer Fieldwork

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