Comparative+Genomics


 * __Methods __**

This source was used to discuss many features of the CFTR gene **.**
 * HapMap **[|http://hapmap.ncbi.nlm.nih.gov]

This source was used to understand phenotypes of the CFTR gene.
 * Ensembl **[]

This source was used to have a visual of both the CFTR protein and one of the mutations.
 * Protein Databank **[]

1. Search for "CFTR" under the gene search on [|NCBI] and choose the search result for //homo sapiens// (Human). 2. Scroll down to the "Genomic regions, transcripts, and products" tab and click on Go to "reference sequence details". 3. Under the "mRNA and Protein(s)" tab, choose the CFTR protein, "NP_00483.3". 4. Click on the "FASTA" link. This is where the protein sequence is located. 5. Click "Send to">File>Create File. 6. Your FASTA file will then be located in your Downloads Folder on your computer.
 * CFTR protein sequence **

1. Go to [|NCBI Protein Blast] and upload your newly downloaded FASTA file of the CFTR protein sequence. (All default settings were used) 2. Click BLAST. <span style="font-family: 'Times New Roman',Times,serif;">3. You will get several blast hits here, 25 top hit species were chosen to align. <span style="font-family: 'Times New Roman',Times,serif;">4. Once all your hits are selected, go to "Download">FASTA (complete sequence)>Continue. <span style="font-family: 'Times New Roman',Times,serif;">5. Your selected species protein sequences will be downloaded in a FASTA file located in your Downloads Folder.
 * <span style="font-family: 'Times New Roman',Times,serif;">Protein BLAST **

//<span style="font-family: Times New Roman,Times,serif;">You will need to download SeaView software for this step which can be located at this link [|SeaView]. // <span style="font-family: 'Times New Roman',Times,serif;">1. Open SeaView and click on File>Open>Your downloaded FASTA file from NCBI Blast. <span style="font-family: 'Times New Roman',Times,serif;">2. Align>Align All>OK. (This step will take less than a minute). //<span style="font-family: 'Times New Roman',Times,serif;">You are now ready to create your three types of trees. // <span style="font-family: 'Times New Roman',Times,serif;">3. Trees>Parsimony. <span style="font-family: 'Times New Roman',Times,serif;">4. Trees>Distance Methods. <span style="font-family: 'Times New Roman',Times,serif;">5. Trees>PhyML. (This step may take up to two minutes). <span style="font-family: Times New Roman,Times,serif;">6. Each tree should be re-rooted to the Weddell Seal which can be done on each tree tab. <span style="font-family: Times New Roman,Times,serif;">-//No bootstrapping methods used to create these trees//
 * <span style="font-family: 'Times New Roman',Times,serif;">Creating Phylogenetic Trees **

//<span style="font-family: Times New Roman,Times,serif;">Have FASTA file open that you downloaded from NCBI BLAST. // <span style="font-family: 'Times New Roman',Times,serif;">1. Go to [|Pfam Search]. <span style="font-family: 'Times New Roman',Times,serif;">2. Copy and paste each protein sequence from your FASTA file for each selected species. This will give you an image of where the CFTR gene is in relation to other genetic elements.
 * <span style="font-family: Times New Roman,Times,serif;">Pfam Domain Data **

//<span style="font-family: 'Times New Roman',Times,serif;">All methods accessed March 2016. //


 * __<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Results-Comparative Genomics of the CFTR Protein __**

<span style="font-family: Times New Roman,Times,serif;">There are many ways to analyze the comparative genomics of any gene. For this analysis, the CFTR protein sequence for humans was blasted on NCBI to find similar protein <span style="font-family: Times New Roman,Times,serif;">sequences in various other species. The protein sequence of the CFTR gene was used in this case due to the fact that the nucleotide sequence was too long to work with. The sequencing data was downloaded as a FASTA file and put into the <span style="font-family: 'Times New Roman',Times,serif;">multiple sequence aligner, <span style="font-family: Times New Roman,Times,serif;">Seaview to be aligned and assembled into a parsimony tree, a distance tree, and a maximum likelihood tree. All trees were rooted to the Weddell Seal.

<span style="font-family: Times New Roman,Times,serif;">In **Figure 5A**, a selected region of amino acids (559-688) were aligned in Seaview for each of the chosen species taxa. From this section, one can see a strong alignment between the selected species. This could also indicate little various between the protein sequence of the CFTR gene.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">
 * <span style="font-family: 'Times New Roman',Times,serif;">Figure 5A. Aligned Protein Sequences at a Selected Region (Amino Acid 559-688) Image retrieved from SeaView **

<span style="font-family: Times New Roman,Times,serif;">After the sequences have been aligned, a parsimony phylogenetic tree was created. The parsimony tree in **<span style="font-family: 'Times New Roman',Times,serif;">Figure 5B **<span style="font-family: Times New Roman,Times,serif;"> represents the preferred hypothesis of relationships among all of the taxa in this tree. The branch lengths are equal to the average number of changes over all sites in the protein sequences. Each new branch is created to show a site that has a most parsimonious reconstruction that would exhibit change in that branch, (Gouy, //et al.,// 2010). This tree shows similarities between like species by grouping them together in their own clades. This tree however is not the best way to represent relationships between the species due to the fact it does not take all variation into account.

<span style="font-family: 'Times New Roman',Times,serif; line-height: 0px; overflow: hidden;">
 * <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;"> Figure 5B. Parsimony Tree for CFTR Protein with Gene Location Regions. (Protein region images retrieved from Pfam). **


 * <span style="font-family: 'Times New Roman',Times,serif;">Figure 5C **<span style="font-family: 'Times New Roman',Times,serif;">is a distance tree that shows variation in the CFTR protein sequence across different time periods. Again, this tree does not take into account all variation within the protein sequence. It is not considered as accurate as the Maximum likelihood tree in **Figure 5D**.


 * <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;">Figure 5C. Distance Tree for CFTR Protein **

<span style="font-family: 'Times New Roman',Times,serif;">The third tree generated in **Figure** 5**D** shows a maximum likelihood tree, the most useful tree for understanding the evolutionary history of this gene.

<span style="font-family: 'Times New Roman',Times,serif;">To the right of the tree, protein location images are present for each species listed. This shows what type of variation occurs with where the CFTR gene is in relation to the different elements around it. It is apparent that there is an ABC transporter downstream and a ABC membrane upstream from the CFTR gene. For the majority of the taxa, these domains line up quite well. It is apparent that there is some variation, for example, in the European Hedgehog where the first ABC membrane begins further along in the sequence than all other species. The hedgehog also seems to have a cut off secondary ABC membrane next to the CFTR gene. These protein images were created by using each protein sequence in the Pfam database.
 * <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;">Figure 5D. Maximum Likelihood Tree for CFTR Protein **

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