Today we will be assembling the mitogenome of a 'species of special concern', Blainville's horned lizard (Phrynosoma blainvillii, Family Phrynosomatidae). Our work will represent the first complete mitochondrial genome to be published for the genus Phrynosoma. Based on a search of Entrez Nucleotide, there are three complete published mitogenome sequences of Phrynosomatidae (Urosaurus nigricaudus- 17,298 base pairs (bp), Sceloporus occidentalis - 17,072 bp, and Uta stansburiana - 16,703 bp). The horned lizard mitogenome is thus estimated to be about 17,000 base bp in length and to contain 2 ribosomal RNA genes, 22 transfer RNA genes, and 13 genes involved in electron transport and oxidative phosphorylation. PowerPoint.
Part 1- Genome assembly using the map to reference function in Geneious
1) Open the Geneious application
2) Drag the "Lizarddata.fasta" file into the large empty box in Geneious
3) Import the reference sequence. We will use the Uta stansburiana sequence cited above. Click on this link here to download the Uta mitogenome. Once you have this file, drag it into the Geneious window just like you did before with the Lizarddata.fasta DNA sequencing file.
4) Select the Uta reference file by clicking on it and then while holding down the shift button also select the Lizarddata.fasta file.
5) Click on the box above that says “Align/Assemble”, scroll down and select “Map to Reference”. A window will open.
6) Under the “Fine Tuning” line, select “Iterate up to 10 times”. On the middle left of the window click the “Do not trim” button. In the middle right click on the “Save contigs” box. Click “OK”. The mapping will take approximately 15 minutes.
Part 2- Genome assembly continued, closing the gaps (if any) and examining the sequence
1) Click on the far left of the screen around position 1, then magnify the reads that were mapped by clicking 7 or 8 times on the “+” lens icon in the upper right of the Geneious screen.
2) You should see Phrynosoma (Horned Lizard) DNA sequences mapped to the Uta reference. Scroll through some of the sequences to see the mapping that Geneious performed.
3) Right click on the word “Consensus” in the upper left of the screen, then select “Copy”. Then paste this sequence into a new Text or Notepad file. At the very top of this file write “>PhrynosomamtDNAdraft1”, then hit return. Save the file as “PhrynosomamtDNAdraft1.fasta” to your desktop or a folder on your computer.
4) Scroll or search to find any ???? or ambiguous letters such as "N" (regions of the sequence that Geneious was unable to map). You can usually correct errors or issues manually. If not, then we can close any questionable gaps if there are any using Geneious. If there are gaps, drag the “PhrynosomamtDNAdraft1.fasta” file into Geneious. We are now going to use our draft as the seed (bait) for a mapping of 5 iterations.
5) Select the bait file (PhrynosomamtDNAdraft1.fasta) and the Lizarddata.fasta file.
6) Click on the box above that says “Align/Assemble”, scroll down and select “Map to Reference”. Use the same settings as before (except =5 iterations), click “OK”.
7) When the mapping is complete, examine the results and see if the bait sequence was extended over all of the ????? If it did not, go to the ????? (region of the sequence that Geneious was unable to map). Select about 150 bp of the sequence to the left of the ?????, then paste this 150 bp into a new Text file. At the very top of the new text file write “>gap1growtoright”, then hit return. Save this file as “gap1growtoright.fasta”. This is your bait or seed file.
8) Drag this bait file ("gap1growtoright.fasta") into Geneious. Select this file and the Lizarddata.fasta file again, then click on the box above that says “Align/Assemble”, scroll down and select “Map to Reference”. Use the same settings as before (=5 iterations), click “OK”.
9) Examine the results. If the program added reads to your bait file, extending to the right (3' direction) over the ????? region, then copy and paste the correct DNA sequence over the ?????? in your PhrynosomamtDNAdraft1.fasta file, thereby closing the gap.
10) If there are other ?????? regions, then perform the same steps above for 4-8. Once you think you have the entire DNA sequence, then save this file. Drag it into Geneious, and confirm that there are no errors in your sequence by mapping one final time. Follow numbers 6-8 above.
Part 3- Aligning the mitogenome sequence of Phrynosoma blainvillii to other closely related lizards
1) Open the mitogenome DNA sequence file of Phrynosoma blainvillii. “Select all” of your assembled DNA sequence and paste it into the input window in MAFFT (http://mafft.cbrc.jp/alignment/server/index.html). Then paste all of the Fasta mitogenome DNA sequences from other closely related lizards into the same window. Get other lizard mitogenome sequences here . Click “Submit” to perform the alignment.
2) When the alignment is complete, click the “Reformat” button. When the new window opens click on “Output sequence format:”, then select “Phylip|Phylip4”, then click on “Download to file”. Save this file to use for the phylogenetic analysis.
Part 4- Phylogenetic analysis of P. blainvillii with other lizard mitogenomes
1) We will use an online site called Trex-online, found here: http://www.trex.uqam.ca/. Go to this site and in the box on the screen paste all of the alignment data into it. Scroll down a little and then click “Compute”.
2) When the results are finished, in about 1 minute, click on “View Tree”.
3) Another method for visualizing the phylogenetic tree is to click on “Best Tree”. Copy the data present, then open Phylogeny.fr: http://phylogeny.lirmm.fr/phylo_cgi/index.cgi
4) Select “Online Programs”, click on “TreeDyn”, and then paste the data into the box and click “Submit”.
5) After this analysis is complete you will need to format the tree by selecting the proper outgroup and swapping some branches.
Part 5- Annotation of the mitogenome
1) Upload the completed fasta formatted DNA sequence of Phrynosoma into MITOS (http://mitos.bioinf.uni-leipzig.de/index.py) by clicking on “Choose File” and then selecting your completed mitogenome assembly. Also fill in the other information they request. Lizards are vertebrates, so make sure the Genetic code is set to 02 (=Vertebrate). Then click “Proceed”. This will take approximately 1 hour - 3 days to complete. Rather than wait, click here to see what the results look like.
2) MITOS does a great job with rRNAs, tRNAs, and most of the genes involved in electron transport and oxidative phosphorylation, but we still need to make corrections for some of the start and stop positions of the genes in our sequence. Using the MITOS link I provided above, we want to download this information by clicking on “TBL file” in the upper left. TBL = Table. Open this file and save it.
3) To make corrections to your annotation file we'll be using Sequin. Sequin can be downloaded here Sequin.
4) To start the Sequin program, double click on the Sequin icon on your desktop, then click on “Start New Submission”.
5) The program will now take you through a series of windows that you populate with information. We will only submit one file to GenBank in the end, with all of the appropriate names and data. What you put in here is just for your experience/training. Enter information in the boxes that Dr. Hughey provides.
While you are working with Sequin, you'll need to have the Uta stansburiana sequence file open so that you can compare the amino acid sequences. As noted above, ignore the MITOS rRNA and tRNAs, but correct the other genes start and stop positions by comparing the Uta start and stop positions to our annotation.
Analysis and visualization of the mitogenome
Genome Maps can be drawn with OGDRAW. You use the GenBank flatfile from Sequin to construct your map, go here: http://ogdraw.mpimp-golm.mpg.de/
NCBI Nucleotide database is here: https://www.ncbi.nlm.nih.gov/nucleotide/
Blast search is here: https://blast.ncbi.nlm.nih.gov/Blast.cgi
Links for writing the Mitochondrial DNA Part B: Resources manuscript
-Gray's Original publication of this species can be found here on page 96
-Sequin file (email Dr. Hughey for this file)