We did not successfully sequence forward and reverse reads of EPIC makers for approximately 25 individuals of Mimulus cardinalis from various locations. Instead of using our own data for the protocol we used the sequences of previous classes. The first section revolved around coding heterozygotes. First a new folder was created and it was labeled New 5525 in the Geneious program. From the file “EPIC Sequence Reads” we downloaded the assigned marker forward and reverse sequences. For our group we downloaded the document for the EPIC marker 5525. Once the files were in Geneious they were all selected and an alignment using Muscle was created. The alignment was opened and trimmed at the ends. Following this step the polymorphisms and heterozygotes were counted and recorded on the space provided on the protocol sheet. Once the process was completed, the IUPAC Ambiguity Code list was used to record the heterozygote sites that were also polymorphisms with the appropriate ambiguity code. Once all the edits were completed the page was edited and the changes were saved.

The second half of the protocol was the assembly of EPIC marker alignments. We created consensus sequences from Forward-Reverse pairs. The two sequences were selected and assembled using the De Novo Assembly feature. All default setting were kept the same and a new file was generated. The new file was trimmed at the ends as well to remove bad regions. The pop-up was closed to save the changes. The steps to create and assembly using the De Novo Assembly and editing steps were repeated for all the Forward-Reverse pairs we download from the file on canvas.  After all the assemblies were generated, all the new files were selected a Consensus Sequence was produced. In the new file were all the consensus sequences which were extracted by using the feature “Extract sequences from the list” along with the “Extract to a new folder called” setting. The out group was also moved into the new file. Once the folder was generated all the files were selected and edited to display only the sequence name as the file name. This was done to prepare for future steps that would be taken using the data.

Finally, we built an alignment by selecting all the sequences and constructing an alignment using Muscle. The end were trimmed and the edits were saved. The name was edited to display the EPIC marker name assigned. At home we were instructed to infer a Bayesian tree for the marker alignments. M. lewisii was set as the out group. The run was set for a long period of time to obtain a good posterior distribution and fuzzy caterpillar. The data collected in each step of the protocol is included below.

Questions:

Polymorphic Columns: 54 (T or A), 62 (T or C), 80 (G and C), 146 (C and G), 197 (A and G), 295 (C and G), 299 (A and C), 334 (A and G), 340 (A and C), 403 (G and A)

Heterozygotes: 92 (C and T), 85 (G), 340 (C and A), 321 (A and C), 197 (A and G)

Heterozygotes that are also polymorphic: 4

Figures:

Bayesian  Phylogenetic Tree:

Analysis of Bayesian Phylogenetic tree:

There are two strongly supported clades. The clades do represent geographic populations.

Posterior Distribution:

Trace: