Genomics Workflow
Before coming to class on March 2, 2018, you need to download 1) Geneious, 2) Sequin, 3) Fucus vesiculosus reference, and 4) the genomic data.
= 10 points
1. Geneious - Go to this link to download a 14 day trial of Geneious: http://www. geneious .com/
Click on "Free Trial" and then enter your information. Use "Hartnell College" for the Institution, and "Student" for the user type. Use your student email. Geneious will send a trial license code to the email you provide. Once you get your license number (16 digit number) via email, you'll need to copy it from the email and paste it into one of the Geneious box after you install the Geneious software. To get the license activated, you need to type your name into the Evaluatee box, and you will also need to paste the entire license, including the word "trial" into the license box, i.e. "Trial_XXXX_XXXX_XXXX_XXXX" to get it to work. Let me know if you have issues.
Typically you have to wait 5 minutes to 1 day for someone from the Geneious team to send you the trial license code, so don't wait until the morning of the workshop.
2. Fucus vesiculosus reference - Click on this link Fucus vesiculosus reference , then click on "Send to:" in the upper right of the screen. A window will pop-up, select the "File" button under Choose Destination. Finally, click on "Create File". The file will download. Save it somewhere on your computer where you can access it.
3. Genomic Data - Check your Hartnell student email, Dr. Hughey will send you the data. IMPORTANT- Do not try to open the data, just download it. We will drag and drop it into the Geneious "Local" folder when we begin.
Summary
This week we will be assembling and annotating the mitogenome of the rockweed Fucus spiralis L. (Family Fucaceae). Our work will represent the first complete mitochondrial genome for this species, and it will be published. Based on a search at Entrez Nucleotide , there are two complete published mitogenome sequences of Fucaceae (Fucus vesiculosus- 36,392 base pairs (bp) and Fucus distichus- 36,400 bp). The F. spiralis mitogenome is estimated to be about the same size, around 36,400 bp in length. Both of these mitogenomes have about 67 genes, and contain the following genes: cob and tatC, 3 rRNA, 3 open reading frames, 3 ATP synthase, 3 cox, 6 rpl, 10 NADH, 11 rps, and 26 tRNA genes (trnL occurs in triplicate and trnI, trnM, trnS, trnY in duplicate). Based on hybridization and DNA studies, F. vesiculosus has been shown to be very closely related to F. spiralis (See Fucus links at the bottom of this screen). In fact several earlier phycologists considered F. spiralis to be a variety of F. vesiculosus. PowerPoint .
Part 1- Genome assembly using the map to reference function in Geneious
= 10 points (Submit to Canvas )
1) Open the Geneious application
2) Drag the "Fucusspiralisdata" file into the large empty box in Geneious
3) Import the reference sequence. We will use the Fucus vesiculosus sequence noted above because of its close evolutionary relationship to F. spiralis. Click on this link to download the F. vesiculosus mitogenome . Once you have this file, drag it into the Geneious window just like you did before with the Fucusspiralisdata.fasta file.
4) Select the Fucus vesiculosus reference file by clicking on it and then while holding down the shift button, also select the Fucusspiralisdata 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 5 times”. On the middle left of the window click the “Do not trim” button. In the middle right click on the “Save contigs” box. Under “Sensitivity” click on the scroll down menu, then select “Low Sensitivity/Fast”. Click “OK”. The mapping will take approximately 5 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 Fucus spiralis DNA sequences below, that were mapped to the Fucus vesiculosus reference above. Scroll through 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”. Paste this sequence into a new Text or Notepad file. At the very top of this file write “>FucusspiralismtDNA”, then hit return. Save the file as “FucusspiralismtDNA.fasta” to your desktop or a folder on your computer.
4) Scroll or search to find any ??? or ambiguous letters such as "N" (N = regions of the sequence that Geneious was unable to map). You can usually correct errors or issues manually. If there are no gaps or issues, then we are done!!!!
Or
If there are issues, then we can fix them or close any questionable gaps, if there are any, using Geneious. If there are gaps, drag the “FucusspiralismtDNA.fasta” file into Geneious. We are now going to use our draft as the seed (bait) for an additional mapping of 5 iterations.
5) Select both the bait file (FucusspiralismtDNA.fasta) and the Fucusspiralisdata.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, click “OK”.
7) When the mapping is complete, examine the results and see if the bait sequence was extended over the ??? If it did not, then go to the ??? (region of the sequence that Geneious was unable to map). Select about 250 bp of the sequence to the left of the ???, then paste this 250 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 Fucusspiralisdata.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), then 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 newly added extension of DNA sequence over the ??? into your FucusspiralismtDNA.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- Annotation of the mitogenome
= 40 points (Submit to Canvas )
1) Go to the Fucus vesiculosus annotation in GenBank . In the upper right, click on “Send To”. Under “Choose Destination”, click on the “File” button. Under “Format”, select “Feature Table”. Click on “Create File”. Drag this created file onto your desktop, or into a folder where you can find it, and open it. We will use this file as a template for defining the start and stop positions of (annotating) our F. spiralis genes. We can do this, because F. spiralis is very closely related to F. vesiculosus. It has the same genes and gene organization.
2) Run a Blast Search using your F. spiralis mitogenome. To do this, copy and paste your entire F. spiralis mitogenome sequence into the “Enter Query Sequence” box at the top of the screen. In the “Organism” rectangle below, type in “Fucus vesiculosus”. Then scroll down to the bottom and click on the “Blast” button.
3) After the alignment is complete, scroll down to the alignment of the two sequences. Remember that the query sequence is our sequence, and the subject sequence is F. vesiculosus. What we need to do is transcribe the nucleotide number from this alignment to the Feature file. We need to do this for each gene, one by one, to ensure that we get the correct start and stop gene positions.
4) After all of the start and stop positions have been entered into your features file, save that file and then open 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 will 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 in class.
Part 4- Aligning the mitogenome sequence of Fucus spiralis to other closely related brown algae
1) Open the mitogenome DNA sequence file of Fucus spiralis. “Select all” of your assembled mitogenome DNA sequence and paste it into the input window in MAFFT . Then paste all of the Fasta formatted mitogenome DNA sequences from other closely related algae into the same window. Get other brown algal mitogenome sequences by performing a Blast Search using your F. spiralis mitogenome sequence. Click “Submit” in MAFFT 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 5- Phylogenetic analysis of Fucus spiralis with other brown algal mitogenomes
1) We will use an online site called Trex-online . 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”.
or
3) Another method for visualizing the phylogenetic tree is to click on “Best Tree”. Copy the data present on the screen, then open Phylogeny.fr
4) Select “Online Programs” in Phylogenyfr., 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.
Additional Links:
Analysis and visualization of the mitogenome
Genome Maps can be drawn with OGDRAW. You use the GenBank flatfile from Sequin to construct your map.
NCBI Nucleotide database is here: https://www.ncbi.nlm.nih.gov/nucleotide/
Blast Search is here: https://blast.ncbi.nlm.nih.gov/Blast.cgi
Writing the Mitochondrial DNA Part B: Resources manuscript
= 40 points (Submit your final draft to Canvas )
-Mitochondrial DNA Part B website
-Example of a Fucus Mitochondrial DNA Part B paper
-Use this Fucus draft Template, revise the red colored font with our F. spiralis data and information, leave the black font
-Tree
- Fucus paper 1, Fucus paper 2, Fucus paper 3, Fucus paper 4, Fucus paper 5, Fucus paper 6