Link to this post | posted 04 May, 2020 21:11
fbaliraine	Thanks Debbie! Working on refining a student's version, needed cross-check whether had been deleted during annotation, but at least I did not see it called in the DNA Master file I am working on. Thanks! Fred

Link to this post | posted 04 May, 2020 16:36

fbaliraine

Dear Phage Hunters, Here is a real weird one for phage MrMiyagi. Not autocalled in DNA Master, no Coding potential seen in GeneMark (smeg), S, or TB. But seen as in phamerator, sharing the same pharm 9086 with gene 31 in phage Fowlmouth. When inserted, shows poor RBS scores (z= 1.353, F =-6.153), but surprisingly, TmHMM & SOSUI analysis clearly show that this is a membrane protein! (See figures below). Blastp yields q11:s1 with Cuke; q16:s1, 100%, 6e-39 with Fowlmouth; no realistic way to get it to be q1:s1 with Cuke of Fowlmouth. HHPred yields no hits above 90% probability [The highest hit, at 83.65% probability (4IMM_B; PDBe ), hits the "Outer membrane assembly lipoprotein YfgL; 8-bladed beta-propeller, Protein-protein interactions, chaperone; 2.33A {Moraxella catarrhalis"]. What’s your take? Edited 04 May, 2020 16:52 313Kb

Link to this post | posted 04 May, 2020 04:53
fbaliraine	I am settling for start #3 in MrMiyagi, which has a better RBS score (Z = 3.053, -2.645)and is present in all members of the pharm, called 2/3 of the annotated genomes so far (see attached). Thanks. 18Kb

Link to this post | posted 04 May, 2020 03:06
fbaliraine	Thank you Debbie! Fred Edited 04 May, 2020 03:07

Link to this post | posted 30 Apr, 2020 04:33

fbaliraine

I am inclined to change a start from that called by GM, in favor of the next one which definitely has a better RBS score, but then this question of curiosity comes to my mind: Often, we annotators will scan the sequence to look for the best RBS score. However, ribosomes are “machines” which I would expect to bind at the earliest strong binding site they can find along their path down the sequence. It is understandable for places with z values below 2, but from the resource guide, “A Z-value higher than 2 is getting good”. If ribosomes do not go back and forth, first taking a “dry run” through the sequence like us humans to choose the best RBS score, what would then make a ribosome to skip start 2 in the attached figure (z= 2.018, Fs -5.536, called by GM in MrMiyagi at position 35962 bp), for example, and go several bases down the road to choose start 3, which has a better RBS score (Z = 3.053, -2.645)? 175Kb

Link to this post | posted 30 Apr, 2020 03:12

fbaliraine

Dear Phage hunters, I am strongly inclined to deleting reverse gene 36 in phage MrMiyagi (SSC: 33325-33185 reverse, RBS z=2.o12, Fs =-5.0722), but perhaps a second opinion might help before I pull the trigger, since this is an orpham with not other data. Here’s the deal. It was called by GM, but not GM. No hits in phagesDb, but Blast via DNA master shows a hit at “extracellular solute-binding protein, NCBI, Roseovarius sp., WP_138933805, 58.70%, e-value 2.24 (13.3% similarity; e-value not significant; https://www.ncbi.nlm.nih.gov/protein/WP_138933805.1/). NB. Roseovarius is a genus of the Rhodobacteraceae. No coding potential in both GeneMark (smeg) & TB, but there is atypical CD in GeneMarkS. This "gene" is in a large gap between forward genes, and I know it is not common for phages to switch between forward and reverse genes. What is your take on this? Is atypical coding potential ever acceptable to be used? see attachment. Thanks! Fred Edited 30 Apr, 2020 03:16 79Kb

Link to this post | posted 26 Apr, 2020 20:40
fbaliraine	Thanks a lot Debbie! This helps a lot. Case closed! Fred

Link to this post | posted 26 Apr, 2020 02:34

fbaliraine

Hi Debbie, Thanks for your response. It is phage Heath. The file is attached. I inserted gp 60, so anything beyond gp 60 will be plus 1bp from the draft sequence. The question is about feature 79 (78 in Draft sequence). I’ve checked, Starterator and realize that it is an Orpham, no data. Now that you have the file, could you kindly as well look at gp 74 (73 in draft), it is also an Orpham. The explanatory notes for available in the notes section. In both cases, it seams to me that GL/GM are is preferring ATG over GTG starts. Thanks! Fred Edited 26 Apr, 2020 02:44 447Kb

Link to this post | posted 25 Apr, 2020 08:55

fbaliraine

Dear Phage hunters, Please help me decide on this start. Dilemma: From BLAST searches, everyone else seems to have selected the start called by both GM and GL. I don’t want to go against the tide but…Whereas the start from 63861 bp (ATG) which was called by both GL & GM gives the best RBS score (Z = 3.35, FS -1.993), it leaves a huge (423 bp) gap, and gives a short (162 bp) ORF, compared to position at 64122 bp (GTG) , which has RBS score (Z= 2.151, Fs -5.229), has a 30bp overlap which is acceptable according to the guiding principles of annotation, and gives a far longer ORF (423 bp vs 162). Thanks! 151Kb

Link to this post | posted 14 Mar, 2019 20:15

fbaliraine

Steven Caruso If you look at the tape measure protein in coliphage HK97: https://www.ncbi.nlm.nih.gov/protein/NP_037710.1. It's a big protein, 1089 amino acids long. I think what HHPred is seeing is your protein looks like a small chunk of it, but clearly you have a much better candidate in gp17 for tape measure. Since it is far past tape measure, and not one of the large genes immediately following it, it's hard to justify using synteny to call it a minor tail protein. Your best HHPred hit is to a DUF, as is your second good match, then human signaling proteins. It would be hard for me to make a call on this one other than NKF. Steve Thank you Steve! Case closed! Fred