Link to this post | posted 05 May, 2020 19:23

fbaliraine

Having trouble deciding on current Genes 42 and 43 in Phage MrMiyagi. Attached, please find snapshots of RBS scores, Frames, Coding potential, and Phamerator maps. These two gens apparently have overlapping ORFs. No CP seen in GeneMark-smeg & TB, but strong coding potential for both in GeneMarkS. Start for gp 43 (45 in pharmerator) called by GL & in Cuke & Fowlmouth; staterator; SSC: 35734; Z= 1.676, F= -6.512; spacer distance 16; with 6 bp overlap; we were selecting the start at 35716, Z= 2.487, F= -5.050, 26 bp overlap with gene 42 (44 in phamerator); with a shorter spacer distance of 5, but realize this gene 43 ORF is continuous from 35449 bp (Z= 2.018, F= -5.536; the next start is with a better RBS at 35572 bp with Z= 2.673, F= -3.504). The short gene 43 (45 in phamerator) apparently currently has only 3 members in the pham 32826; compared to 44 which has 61 members (pharm 40615). Start for gene 42 (44 in phamerator) is inside CP plateau, hence not covering all CP, but it’s the 1st one; see CP figure attached. Since there is no break/stop codon in the ORF for gene 43(45 in phamerator) and there’s strong RBS scores for the 1st and 3rd starts which are overlapping gene 42 (44 in phamerator) is there no chance that this is the same gene? Should I delete 42 (44 in phamerator) and take the start at 35572 bp with Z= 2.673, F= -3.504, or should I just overlook this, keep gene 42 (44 in phamerator) and take the start called by GL at 35734; z= 1.676, F= -6.512 and in Cuke & Fowlmouth 6bp overlap, or the one I am considering with a better RBS score at 35716, Z= 2.487, F= -5.050, 26 bp overlap? Please advise. Edited 06 May, 2020 16:22 734Kb

Link to this post | posted 05 May, 2020 16:29
fbaliraine	Great insight! Thanks Debbie! Fred

Link to this post | posted 05 May, 2020 02:03
fbaliraine	This is the last one on MrMiyagi. Inclined to delete but seeking 2nd opinion 1st. Feature 117 SSC: 67883-68098. Orpham, no Starterator data. #119 on pharmerator fig attached. CP not seen in Genemark (smeg) & TB, but strong CP in GeneMarkS (fig attached). No significant blast search results. Your take? 226Kb

Link to this post | posted 04 May, 2020 21:18
fbaliraine	Probably RNA polymerase sigma factor? I've just been looking at the HHPred data for the Reference sequence Nerujay_52 provided in the Official Functions list and this is what I see: https://toolkit.tuebingen.mpg.de/jobs/Nurujay_Gp52 (also attached for comparison) Fred Edited 04 May, 2020 21:20 57Kb

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