TITLE: Finalizing Mitogenome Annotation in Geneious
AUTHORS: Dakota Betz

[DESCRIPTION]
Our protocols are constantly evolving and old versions will be deleted.
The documents here are not intended to be cited in publications

[STEPS]
SECTION: Opening Geneious and Setting Up Workspace
2. Log into Geneious - (or, if you have your own version, just open that) Use the floating license server. IT IS VERY IMPORTANT YOU DO NOT UPDATE GENEIOUS - the floating license will not work with newer versions.
SECTION: Importing Sequences
3. Importing Your Data
SECTION: Importing Sequences
3.1. Drag In Your "...contig.fasta" File: Go to wherever you have your assembled mitogenome in Finder. Expand the MitoFinder folder, then expand the "..._Final_Results" folder, and find the "Sample_MitoFinder_100p_mtDNA_contig.fasta" file.
 

SECTION: Importing Sequences
3.2. Drag the "Sample_MitoFinder_100p_mtDNA_contig.fasta" file into Geneious, where it says "Drop files here to import". Your file should show up like this. If you accidentally placed it in the wrong folder, you can click on it and move it around, like in Finder.
 

SECTION: Importing Sequences
3.4. Circularization: The mitogenome should be circular. However, MitoFinder most often does not find the circularization and leaves your genes linear. If you want to check, locate the "Sample_MitoFinder_100p.infos" file and open it in a text editor.
 
If circularization is not found (which you can also see by seeing Geneious does not automatically circularize it), we want to manually force it. The non-coding region (D-loop) can be very repetitive and it's hard for the program to decide where the genome ends. 
SECTION: Importing Sequences
3.5. Forcing Circularization: Right click on your sequence in Geneious and click on "Circularize Sequence".
 

 
The change should show up in your bottom part of Geneious, where the sequence is no longer mapped in a straight line, but now becomes a circle.
 
Save changes.

 

SECTION: Importing Sequences
3.3. Drag In Your "contig.gff" File: In the same folder as your "contig.fasta" file, locate the "Sample_MitoFinder_100p_mtDNA_contig.gff" file, and drag it into Geneious, into the same area where your "contig.fasta" file shows up. This will visually map the genes MitoFinder found.

Sometimes Geneious will ask you questions about this - i.e. what you want to map it to, just say yes for everything.
Sometimes after importing your data won't visually look like this - usually, deleting the files from your Geneious working folder and re-importing them fixes this issue (sometimes a you have to re-import multiple times before it works)
 
SECTION: Opening Geneious and Setting Up Workspace
2.1. When prompted, enter the following server IP address and port, and make sure to select "Use floating license server".

 

 
 
SECTION: Opening Geneious and Setting Up Workspace
2.2. Create A New Folder: When you first open Geneious, you should have a blank workspace with some sample documents.

 
You will want to create a new folder for the mitogenome you will work on. To do this, go to the tool bar at the top of your monitor and click "File", then "New", and "Folder..."
 
SECTION: Opening Geneious and Setting Up Workspace
2.3. Name it something like "SpecimenFamily_Mitogenomes" (i.e. "Pilargidae_Mitogenomes"), or whatever is useful to you. It's good practice to not use any spaces in the folder and file names.
SECTION: Opening Geneious and Setting Up Workspace
2.4. If you will be working on multiple specimens in the same family or greater taxonomic category than just one sample from one species, make another folder within the folder you just made i.e. with your specific sample name.

 

SECTION: Importing Sequences
3.6. Import Amino Acids and Nucleotides: In Finder, in the same folder as before, locate and drag into Geneious the "Sample_MitoFinder_100p_mtDNA_contig_genes_AA.fasta" and the "Sample_MitoFinder_100p_mtDNA_contig_genes_NT.fasta" files. You don't want to import the "..._final_genes_..." files, because they aren't necessarily correct.
 

SECTION: Importing Sequences
3.7. Geneious will ask how you want the sequences from the files stored. Choose "Keep sequences separate".
 
Your Geneious workspace should then look something like this:
 
SECTION: Importing Sequences
3.8. There should be 13 AA files, which are noted by the blue icons - these are the Amino Acid translations of the protein coding genes, and 15 NT files, which are noted by the orange double helix icons - these are the NucleoTide sequences of both the protein coding genes and the non-coding genes (12S/rrnS and 16S/rrnL).

HOWEVER, sometimes MitoFinder doesn't find all of the genes - that's okay; that's why we've run MITOS, and if even MITOS doesn't find them, then we can troubleshoot using steps outlined later on in the protocol.
SECTION: First Pass Check
4. The first way we will check how correct MitoFinder is, is by checking for start and stop codons in the AA sequences of the protein coding genes.
SECTION: First Pass Check
4.1. Click on one of your AA files (blue icon). In the bottom part of Geneious, you should see a sequence of letters, which are the single letter codes for amino acids. Ideally, the sequence will start with the letter 'M', which signifies a start codon, and end with an asterisk '*', which signifies the stop codon.
 
SECTION: First Pass Check
4.3. Do this for EACH AA file. It is very likely that many of your genes will not start and end with start and stop codons. This is how I like to write my notes, but organize your own however is most helpful to you:

 

SECTION: First Pass Check
4.2. Wherever you are keeping your notes, write down for each gene if it did or did not start with an M and end with an asterisk.
SECTION: Obtaining Reference Mitogenomes for Troubleshooting, if all 15 genes are not present
11. Obtaining Reference Mitogenomes: When manually fixing genes, we help ourselves with reference mitogenomes. We want to import and simultaneously look at our new mitogenome and already published mitogenomes, because those help us make informed decisions when it comes to expanding or shortening a gene.
SECTION: Obtaining Reference Mitogenomes for Troubleshooting, if all 15 genes are not present
11.1. Go to GenBank and search for the mitogenomes of closely related species by typing in the following:
 
Make sure to replace "SampleFamily" with whatever your family of organisms is, so you would search for i.e. " "Pilargidae" & "mitogenome" ". Include the quotation marks, because that means GenBank will show you only exact matches.
SECTION: Obtaining Reference Mitogenomes for Troubleshooting, if all 15 genes are not present
11.2. If your search gives no results, try the searching by your sample's suborder or order, etc. until you get results. You can use WoRMS to help you.
SECTION: Obtaining Reference Mitogenomes for Troubleshooting, if all 15 genes are not present
11.3. Once you have the results, select all of them by clicking the boxes before them and choose "Send to", select "Gene Features", keep the default "FASTA Nucleotide" as the format, and click "Create File".
 

SECTION: Obtaining Reference Mitogenomes for Troubleshooting, if all 15 genes are not present
11.4. Rename the file accordingly and move it into your sample's folder. Then, drag it into Geneious, again keeping the sequences separate. A lot of new NT files should appear in your workspace, making it look like this:
 

SECTION: Obtaining Reference Mitogenomes for Troubleshooting, if all 15 genes are not present
11.5. Sometimes, the uploaded mitogenomes won't have rrnS/12S and rrnL/16S included in the FASTA download. In those cases, you'll have to specifically search for both rrnS and rrnL FASTA files on GenBank of each of the mitogenomes you imported. Download, rename, and drag in those files, too, if you don't already have them.
SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12. The first pass check gave us a general idea of how complete our genes are. Now, we want to carefully check each one (even those that do start and stop with start and stop codons!) and fix those that need fixing.
SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.1. Aligning the Sequences: Start by typing "COX1" into the search bar in Geneious. Then, select all of the COI sequences that were uploaded from GenBank. You want to make sure those are the only sequences selected - you can check this by looking at the upper right hand corner below the search bar (I've noted it with the arrow) - the number of selected sequences should be the same as the number of imported mitogenomes.
 

 


SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.2. Aligning the Sequences: Then click "Align/Assemble" in the tool bar, and choose "Map to Reference...".
 
This window will pop up:
 

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.3. Aligning the Sequences: To specify the reference sequence, which we want to be your sample, click "Choose..." and navigate to your circularized file (green circle icon), then click "Select".

 

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.4. Aligning the Sequences: The rest of the default options are fine, so click "OK". Once Geneious is done, clear the search, and at the top of your files, you should see an alignment (orange icon of three parallel lines). Your workspace should look like this:
 
In the bottom part of Geneious, you now have the alignment. The black bars are where the GenBank sequences aligned with your sequences that MitoFinder found, which are the green and yellow bars.

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.5. Check the Beginning of the Gene: First zoom in to the front of your gene. Do this by clicking on the house icon on the bottom right side of Geneious. Then, place your cursor close to the beginning of the gene you are looking at and click on the two blue arrows pointing towards each other. You can zoom out by clicking on the same icon.
 
This will give you such a view:
 
The invertebrate start codons are ATG and ATA. However, just because MitoFinder decided your gene starts with a start codon, this DOES NOT MEAN that this is the FIRST start codon - make sure to check to the left of the gene to see if there are any more ATG/ATA codons. Take care to go by three nucleotides at a time, otherwise you shift the frame of translation.

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.6. Editing the Beginning: If you found that the current way MitoFinder annotated the beginning is incorrect (either no start codon or not the right one), click on the green and yellow bars and drag them one at a time to the nucleotide they should really start with. Make sure you extend/shorten the gene only by a number of nucleotides divisible by three!

 

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.8. Check the End of the Gene: Either zoom out and place your cursor at the end of the gene and zoom in again, or just scroll to the end of the gene.
The stop codons you are looking for are TAA or TAG. Sometimes, when MitoFinder finds the stop codon, your sequence will end like the others you are aligning it to, and other times it won't. However, it can still be correct, like this:
 
There's no obvious way to change this sequence, it doesn't align with the other reference sequences, but there is a stop codon, so I left it as is.

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.7. Editing the Beginning Example:
MitoFinder didn't find the start codon. When I zoomed into the beginning of the gene, I began to look for start codons. I actually found six potential start codons (underlined red)! However, the one farthest to the left is the correct option.
 
 
I then dragged the gene annotations so they now look like this when corrected:
 
I made sure the amount of nucleotides I added was divisible by three.
SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.9. Editing the End: If you found that the current way MitoFinder annotated the end is incorrect (no stop codon), click on the green and yellow bars and drag them one at a time to the nucleotide they should really end with. Make sure you extend/shorten the gene by a number of nucleotides divisible by three!

 

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.10. Editing the End Example:
MitoFinder didn't find the stop codon. When I zoomed into the end of the gene, I found two:
 
HOWEVER! Stop codons are different than start codons. There can't be a stop codon somewhere within the gene, so even if there is another potential stop codon farther to the right, that is not a stop codon for this gene. You have to choose the stop codon closest to the end of the gene you're fixing. Therefore, I edited this gene like this:
 
I made sure I extended the gene by a number of nucleotides divisible by three - in this case 45.
SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.11. I Can't Find a Stop Codon?
AA residue thing
SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.12. Checking the Validity of Edits: After you've edited the annotations of a protein coding gene, there is one more way to check that you have done so correctly. This makes sure you've edited the nucleotides comprising the gene in numbers divisible by three, and also ensures you didn't accidentally add any extra stop codons anywhere.

First, click on the green annotation (the gene), and then click 'Extract'.
 
You can add something to the end of the name, i.e. "...AnnotationCheck" so that you know where the sequence came from later on, then click "OK".

SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
12.13. Checking the Validity of Edits: Then click on the sequence it gives you as an output and click on the "Translate" button. Change the Genetic code to "Invertebrate Mitochondrial", leave the translation frame as 1, and make sure to UNCHECK both of the checked boxes - we don't want to force Geneious to translate the first three nucleotides as a start codon and we don't want it to remove the final stop codon.
 

SECTION: ∂
1. BEFORE you start annotating your mitogenomes, you want to run them through another annotating software, MITOS.
SECTION: ∂
1.1.  Go to the MITOS web server: http://mitos.bioinf.uni-leipzig.de/index.py and upload your sequence.
Enter your name, email, if you want you can add a job identifier (i.e. what is the mitogenome you're uploading for annotation), MAKE SUR·E to change the genetic code to the appropriate choice (in our lab it's generally always "05 - Invertebrate"), then navigate to your assembled mitogenome in Finder. Expand the MitoFinder folder, then expand the "..._Final_Results" folder, and find the "Sample_MitoFinder_100p_mtDNA_contig.fasta" file

 
 

SECTION: Opening Geneious and Setting Up Workspace
2.5. Create another folder within the family or greater taxonomic category folder named "Genes", and within that folder, create 28 new folders. For each protein coding gene, you want 2 folders; one that is "ProteinCodingGene_AA" for the amino acid translation sequence, and the other that is "ProteinCodingGene_NT" for the nucleotide sequence (i.e. ATP6_AA and ATP6_NT). 12S (rrnS) and 16S (rrnL) are not protein coding genes, so for them, you only need one folder each: "rrnS_NT" and "rrnL_NT".

 
 

SECTION: First Pass Check
4.4. Now put away these AA and NT files into their corresponding folders within the "Genes" folder you made. For extra clarity, you can select all of them, 
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
5. 
This is the default way you should do mitogenome annotation. If neither MitoFinder nor MITOS found all 15 genes, then you would refer to "Obtaining Reference Mitogenomes for Troubleshooting, if all 15 genes are not present" onward.
SECTION: Manually Checking and Fixing Protein Coding Genes when all 15 genes are not present
13. 
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
10. 
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
6.5. Force circularize for this sequence, too, like before for the MitoFinder sequence.
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
6.4. Rename the sequence so you know it's from MITOS (i.e. change "MitoFinder_100p.1" to "MITOS").



SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
6.3. Then, drag in the MITOS "___.gff" file.
It might say this:
 
If so, tell it to use what it found, and click "OK".

It might also say this:
 
Select "Continue".

This is what your Geneious should now look like:

 

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
6.2. In Geneious, once again drag in the SAME  "...conting.fasta" file as before.
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
6.1. Hopefully by now, MITOS should have gotten results back to you.

By clicking the links on the left, download the GFF file, FAS file, and the protein plot. Put them in a new corresponding file somewhere that makes sense to you (i.e. a "MITOS" file where you have the FitoFinder file).
 

Scroll to the bottom of the page. If MITOS found anything unusual, it will tell you here. This is an example:
 

Sometimes MITOS will find 2 or more of a gene, or not find some, and that's when the protein plot comes in handy.

This is an example of a zoomed in protein plot:
 

Notice, how under the pink line (nad5), there are 3 other lines, orange, blue and purple (atp8, nad2, and nad4). Sometimes, some genes have certain parts that could be similar, but not very, so it still picks up some signal. The line that has the highest plateau is the one you want to trust. Sometimes, when there are say, 2 atp8 genes that MITOS found, what happened is that the low orange line was found somewhere where there was no other strong signal line above it, but it was still just as low as it is here. Then, you would get 2. But if you checked the protein plot, you would know which one is correct - the one that reaches the highest on the graph.

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
6. Importing MITOS results
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
9. 
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
7. Align the MitoFinder and MITOS sequences for manual annotation and comparison!
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8. Now, it's time to scroll through the mitogenome and manually annotate it based on comparing it with MITOS.
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
7.1. Select both the MitoFinder and MITOS files. Then click "Align/Assemble".

 

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
7.2. Choose "Pairwise Align...", and choose "OK".

 
 


SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
7.3. Your Geneious workspace should look like this:

 

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8.1. Place your cursor at the front of the alignment (click there), and press the zoom in button.

 

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8.2. Scroll to the right until you hit your first annotation. Once you deal with that one, keep scrolling until the next one, and then the next one, etc. until you get through your entire mitogenome!
SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8.3. If it's a tRNA, just check if the MITOS and MitoFinder annotations are the same (if they start and stop with the same nucleotide).
Also check for overlapping - you don't want them to overlap either ith eachother or with other genes. Once you make sure the gene a tRNA is overlapping with is correct, shorten the MitoFinder tRNA annotation so they don't overlap, by clicking and dragging the pink and green bars (each separately) until they don't.
 

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8.4. If it's a protein coding gene or rRNA, stop before the beginning. These are the genes you notes whether they start with an M (the start codon) and end with an asterisk (the stop codon).
First, I like to turn on the Translation - it helps me visually.
1. Go to the second right sidebar tab that looks like a screen
2. Check the box by "Translation"
3. MAKE SURE to change the "Genetic Code" to whatever is relevant for you - in our lab, that's mostly "Invertebrate Mitochondrial"

 
You can check the translation of a part of sequence by clicking where you want to start checking and then dragging to where you want to see - but MAKE SURE you drag from left to right! Drag direction matters!
 


SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8.5. For annotation, you are changing ONLY the MitoFinder sequence, but you are looking at MITOS to help you.

A guiding principle is that you want MINIMAL OVERLAP between genes - if you can entirely avoid it, do. (But at least in annelids, ND4 and ND4L will always overlap ~10 bp!)

To change a MitoFinder annotation, click on the front or back end of the specific green gene bar, and drag it to where you want it (this may take a bit of practice to click correctly for dragging and not just selecting). Then, do the same for the specific yellow CDS bar (coding sequence) or red rrna bar (16S and 12S), and drag that to where you want it, as well.


 

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8.6. At the beginning of a gene, look for the start codon M - the corresponding nucleotides are ATA or ATG.

(Rarely, it could be another set of nucelotides. For Nephytidae, I've run into COI starting with GTG instead).

a.) If the gene starts with an M and the MITOS annotation is either the same, or starts further to the right, keep the MitoFinder annotation as is.

b.) If the gene starts with an M but the MITOS annotation is further to the left and either the MITOS annotation starts with an M or there is an M even further to the left that does not overlap with other genes, change the MitoFinder annotation to whatever the furthest to the left non-overlapping start codon (M) would be.

c.) If the gene does not start with an M, but MITOS' annotation does start with an M, either to the right or to the left, change the MitoFinder annotation to what MITOS found.

d.) If neither MitoFinder nor MITOS annotations start with an M: search for one. Start with the left and go to the right if no luck on the left. Compare gene length to other known genes of the taxonomic group (i.e. if a gene is usually 1500 bp, you'll know something is weird if your gene has 800 bp.).

Below are some examples of editing the beginning of a gene. Feel free to add your own if you don't think they fit into one of the already added examples!
 

SECTION: Manually Fixing Mitogenome Annotations with MITOS (default)
8.7. At the end of a gene, look for the stop codon * - the corresponding nucleotides are TAA or TAG.