Biology 107 Phage genome annotation progressionLesson 1: Learn about your phage and compare its potential genes with similarphageIn this drill you will simply get to know a little bit about the phage whose genome you will be annotating, as well as getting some exposure to some of the tools you will use to begin the annotation process. Some of the steps you do in this drill today you will probably do frequentlythroughout the semester as you carefully inspect each gene call in the section of the genome for which you are responsible.Get to know your phage- Look up your phage by name on the Phages data base(PhagesDB) page and read the information the discoverystudent wrote (https://phagesdb.org/)... Bookmark thispage.- Some useful things you could write in your notebook this week: What species of bacteria does your phage infect (Isolation Host)? When and where was it found?Find a phage with a similar genome sequence- On the PhagesDB page use the green Locally Blast thisgenome- Select the BLAST button and wait for the data to show up…- Your phage is the black line, and also the top red line… the other red lines are other phages similar to your phage, but with different alignment scores- Click on any red line or scroll down to the list of phage below (ordered by alignment score). The higher the score, the closer the alignment)- Click on any score to see the actual nucleotide alignment between your phage and another (be sure to note whichphages are most closely alignedto your phage… that will beuseful later)See where prediction programs think there are protein-coding genes in your phage genome- Sign in to PECAAN (https://discover.kbrinsgd.org/)... Bookmark this page.- In the Summary tab, start typing in your phage name, then select your phage (with the associated lab time) when the name appears.- Select the Genes tab at the top- Select the Host-Trained GeneMark button (GeneMarkpredicts coding potential… the likelihood of a stretch ofDNA sequence could be encoding a protein)- A GeneMark document will appear, and you can scroll horizontally to look along your entire phage genome. Note that there are actually 6 lines, the top three are labeled “Direct Sequence”, the bottom three are labeled “Complementary Sequence” each line represents each coding frame of the DNA, 3 forward frames, 3 reverse frames.Compare the synteny (localization of predicted genes) of your phage genome sequence to a closely related phage- Go back to PECAAN and select the Pham Maps tab - This shows the locations ofpredicted genes (coloredboxes with numbers) andpossible tRNAs (+ signs)- Click and drag or use arrowbuttons to look along thegenomeWhat do the numbers mean? The numbers in the boxes just represent the order in which each gene in that genome appears, starting at the “front end” of the genome. Adding a gene or removing a gene will change the numbering of all of the other genes after it. The numbers along the black, hashed line in the middle represent the nucleotide numbers in kilobases, where the “1” represents 1000 nucleotides/bases fromthe “front end” of the genome.What do the colors mean? If your gene calls are similar in sequence to genes found in other phages, then your gene is considered to be a member of that group of genes (called a “pham”). It means that some version of this gene is known to exist (to some extent) in other phages. If you see a gene box that is white, it means that the predicted gene is unique (it has not been found before in another phage… called an “orpham”)What if all of my boxes are all white, with no colors… are all my genes orphams? No, sometimes there is a delay between the time a phage genome is sequenced and when it is run through a program called Phamerator, which assigns suspected genes to phams. PECAAN is updated regularly, but a newly sequenced genome (like some of ours), will not have gene calls assigned to phams right away. Eventually they will be, soat some point in the semester you should see some, if not all, of the genes in your phageget assigned to a pham (and become a new color).- You can compare the gene calls to the known genes ofother phages that are closely related by using thedropdown menu to select another phage… try selecting a phage to which your phage was closely related in the BLAST search on the PhagesDB (from above, when you checked the BLAST search for similar phages).- This shows you the alignment of your predicted genes with the genes of another phage- Do you see any large gaps? There mightbe a gene in there that wasn’t called andshould be added.- Do you see overlaps of genes? This maymean a gene was called that should notbe there, or the start sight is in the wrong
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