We conducted a double digest restriction associated DNA (ddRAD) study on Mimulus guttatus.  The 1st step was collecting samples which was done on two field trips that can be read: https://usfblogs.usfca.edu/mkmin/wp-admin/post.php?post=23&action=edit. 

Next, we extracted DNA from the samples collected as well as samples previously collected by the graduate student, Alex: https://usfblogs.usfca.edu/mkmin/wp-admin/post.php?post=23&action=edit. 

Next, we double digested our DNA using two restriction enzymes:  https://usfblogs.usfca.edu/mkmin/wp-admin/post.php?post=23&action=edit.  These enzymes cut out the genome into many pieces. 

The unique DNA barcodes were ligated onto each of the individuals.  PCR was used to add a 2nd unique barcode and test if the library construction was successful.  Our PCR was successful, which was photographed by Professor Paul.  After the test PCR, a larger reaction was conducted  (assumed it was successful since 1st one worked).  This was the last step we were able to do as a class.

The next step would be size selection which selects DNA of specific sizes, specifically, we would target from 400-600 bp fragments to use with the Illumina technology.  Size selection can be done in 3 ways by using an automated system called Pippinprep of which we have one in the Suni Lab.   Another way is to use gel extraction (run out full product of PCR and cut out) or use magnetic beads to isolate the DNA fragments.  After size selection, we would then normalize our DNA samples which brings DNA samples to approximately the same concentration.  The number of DNA fragments will be sequenced.  Finally, the last step would be to combine the size selected and normalized PCR products into one vessel.  Then, we would run these samples on any Illumina sequencer (iSeq 1000).  Sequencing would take about 16 hours and if successful, would generate tens of millions reads of the data.  These data would be thrown into a bioinformatics pipeline.  Ultimately, we would align the sequence data with the published Mimulus guttatus genome and call SNPs.  The SNP’s would be used to infer population differentiation using a metric-like FST and access population genetic diversity, such as looking at the number of alleles, allelic diversity, etc.  Based on what I know about Mimulus guttaus, I would expect populations that are geographically divergent to also be genetically divergent.