Link to this post | posted 30 Jan, 2024 19:30

fbaliraine

Thank you, Lee. Indeed, we pointed out the differences to our students and instructed them on what to do, though some still get confused by the mismatches. Since the forward frames in DNA Master perfectly match with the forward frames in the GeneMark coding potential outputs, we were just hoping that there was some way to twitch the GeneMark software for the reverse frames to match those in DNA Master as well. For now, we will just keep pointing out this difference between DNA Master frames and the GeneMark outputs with regard to the reverse frames. Fred Edited 30 Jan, 2024 19:31

Link to this post | posted 26 Jan, 2024 02:19

fbaliraine

As of Jan 25, 2024, whereas the forward frames are Okay, we have noticed a mismatch between the reverse frames in DNA Master and those of GeneMark outputs. We have noticed that that coding potential (CP) for DNA Master frame -1 is in frame -2; plots/data for frame -2 is in the -1 frame, and plots for frame -3 is in the -2 frame of the GeneMark_S, GeneMark_smeg, & GeneMark_TB plots obtained from phagesDB. Is there a glitch or are we missing something? See attached. Thanks! Fred Edited 26 Jan, 2024 02:20 437Kb

Link to this post | posted 21 Jan, 2024 07:58
fbaliraine	Thank you, Debbie! Case closed! Fred

Link to this post | posted 20 Jan, 2024 08:29

fbaliraine

I don’t like the fact that I see no coding potential at FreddyB (subcluster F1) position 44340-44384 bp (MNTYRIPNPVEATQZ) and the fact that it would be the shortest gene I had ever seen. However, this potential gene would be part of an operon with 4 bp (ATGA) overlaps with both the upstream and downstream genes. Given Guiding Principle #12a, the ribosome prefers a 1bp or 4 bp overlap, irrespective of RBS score. I wonder how a ribosome would skip over these two 4 bp ATGA overlaps to exclude this potential short gene that would make part of an operon. BLASTp shows more than 20 hits, with q2:S108, 100% identity, e-value = 0.43. Thoughts? See attached: 328Kb

Link to this post | posted 21 Dec, 2023 04:52
fbaliraine	Thank you! In fact, a look at the six-frame translation shows 3 stop codons between 37305-37388 bp (see attached file). I will go ahead and NOT insert a gene for phage Sonah at position 37305-37388 bp. Fred Edited 20 Jan, 2024 04:45 143Kb

Link to this post | posted 18 Dec, 2023 06:42
fbaliraine	Thank you Debbie! Fred Edited 18 Dec, 2023 18:57

Link to this post | posted 18 Dec, 2023 06:32

fbaliraine

I am asking this clarification question for subcluster F1 and P1 phages. I notice that for phage Sonah (subcluster P1) position 37305-37388 bp (sequence MTDFLGATIRIVAQIGFPTVNPIEVMRZ) has a potential small gene. When inserted and blasted, it gives significant q1:s1, 100% but with just a few phages {Zilizebeth gp 64 (P1), HUHilltop gp 56 (P1) and Royals2015 gp 70 (F1)}, along with insignificant hits with Malithi gp 54 (P1), Camster gp 55 (P1) & Techage gp 59 (P1). I also note that all of the above genes are significantly loger that this potential gene, which would potentially only form part of the fisrt few aa of the above longer genes. I note that a vast majority of previously annotated phages skipped calling this gene. There is no coding potential whatsoever in GeneMarkS, smeg or TB among the above P1 phages that gave singificant hits. The only exception is in the subcluster F1 phage Royals2015 which also has no coding potential in Smeg or TB but has weak/insignificant CP (below 50%) in GeneMarkS (see attached). I note though, that the RBS score for this start in phage Sonah is strong (Z = 2.098; spacer distance = 10; final score = -4.661, with a TTG start codon). Moreover, it has an 8 bp overlap with the upstream gene and would form part of an operon with a 1 bp overlap (TAATG) with the downstream gene. Because this potential gene has weak CP and is only in GeneMarkS of F1 phage Royals2015 among all the genomes with significant hits that I have checked, and has not been called in many previously published genomes, I would like a second opinion about it going forward. Thanks! Fred Edited 20 Jan, 2024 05:37 147Kb

Link to this post | posted 15 Dec, 2023 22:40

fbaliraine

We are dealing with the exact situation with Sonah gp 28 (25227-25475 bp; MWTLKFWKDASERAVKSAAQAAILALGGEAFNAWTVDWQTVGGIALGGAALSLLTSLGSDLLPFGTKGTASLAKLDGEGSARZ). This gene is identical to Langerak gp 28. It is downstream of lysin A & B. DeepTMHMM shows 2 transmebrane domains, and we see the same holin hit in HHPred in PFAM & UniProtKB. Using DeepTmHMM, we note that Langerak gp 29, the gene immediately downstream of Langerak gp 28, is a membrane protein, with one transmebrane domain, just like the downstream gp 29 of Sonah. Even syntenically, I want to call it a holin, but there's again this caveat in the Official Functions list, stating that, "to call a holin…at least 2 transmembrane domains found and the gene be adjacent to the endolysins (s), conserved domain hits (4), and the abscence of additional transmembrane domains in the area." In previous P1 annotations where we relied on the less sensitive SOSUI which classified it as soluble, and TmmHm which shows 0 predicted TMH, and the downstream gene not detected as a membrane proten by these less sensitive software, we had simply called NKF. Now, using the more sensitive Using DeepTmHMM, and given that everything else has been met to call this gene a holin, what do we do about the fact that the immediate downstream gene is actually a membrane protein with one transmembrane domain? — in view of the caveate…the abscence of additional transmembrane domains in the area. Edited 18 Dec, 2023 03:02

Link to this post | posted 10 Jun, 2023 05:47

fbaliraine

I am inclined to call a phosphoesterase function for subcluster M1 phages Glaske16_gp129 (66640-67299) & Dulcita gp 126 (66622-66181 bp), but will seek clarification given that the top 15 hits in phagesDB are to metallophosphoesterases. The sequence is 100% identical between the above phages: MSNVFFTSDLHIGHKKVVASRTTVDGEPAFPDLDNLPEWFGDFEIESYNRILADKWDTTVGKDDVVWVLGDISSGTKSGQEMALEWLSRRPGRKRLIKGNHDGVHPMYRDKAKWVKAYGEVFEDMDTAARIRVALSGGGHVDALLSHFPYMGDHTSVDRHTQWRLPNNGTILLHGHTHSSRRMSSCGGSLQVHVGVDAWNGYPVSMDEIRSYVEIWEDVZ According to the Official SEA-PHAGES Function List, a metallophosphoesterase, "Must contain a HEXXH motif to coordinate the metal ion." I can see the HEXXH motif in Luchodor_gp60, the sample gene for metallophosphoesterase in the Functions list. Whereas there are HHPred hits to metallophosphoesterase, my reasons for being weary about calling it a metallophosphoesterase rather than a phosphoesterase are two-fold: 1. I do not seem to see the HEXXH motif (except if the rule is flexible) 2. The top two HHPred hits are to phages D29 gp 66 & L5 gp 66, both of which are phosphoesterase. D29 is a well-studied, prototypic phage. According to Rudner, Fawcett & Losick (1999; https://www.pnas.org/doi/10.1073/pnas.96.26.14765), the conserved sequence HEXXH is a hallmark of metalloproteases. An HEXXH motif should have an "H" followed by an "E", then by any two amino acids followed by an "H" (see attached). I did a search for "HE" in each of the sequences. >phageD29_gp66 (phosphoesterase) HEXXH motif not seen MSNVWFTSDLHIGHAKVAEDRDWAGPDHDLHLAELWDEQVGKEDVVWILGDISSGGTRAQLDALGWLLNRPGRKRLILGNHDRPHPMYRDAPRLSRLYWNVLDYMSTAARLRVPLDGGGHTNVLLSHFPYVGDHTAEQRFTQWRLRDEGLILLHGHTHSRIIRSTMTNPRQIHVGLDAWHDLVPMDEVREMVNDIEEGL >Glaske16_gp129 (66640-67299) & Dulcita gp 126 (66622-66181 bp); HEXXH motif not seen MSNVFFTSDLHIGHKKVVASRTTVDGEPAFPDLDNLPEWFGDFEIESYNRILADKWDTTVGKDDVVWVLGDISSGTKSGQEMALEWLSRRPGRKRLIKGNHDGVHPMYRDKAKWVKAYGEVFEDMDTAARIRVALSGGGHVDALLSHFPYMGDHTSVDRHTQWRLPNNGTILLHGHTHSSRRMSSCGGSLQVHVGVDAWNGYPVSMDEIRSYVEIWEDVZ Luchodor_gp60 (metallophosphoesterase ref sequence) has the HEXXH motif (underlined) MSKRIVVVSDTQIPFDDRKALKAVVGFIGDTQPDEVVHIGDLMDYPSPSRWTKGTAEEFAQRIKPDSEQAKRRFLEPLRARYDGPVKVHEGNHDSRPFEYLHKFAPALVEYADQFRFQNLLDFDGFGVEVAPEFYKLAPGWVSTHGHRGGVRLTQKAGDTAYNAMMRFNTSVIIGHTHRQGLKPHTLGYGGHQKVLWSMEVGNLMNMHLAQYLKGATANWQTGFGLLTVDGHHVKPELVPVVGGSFSVDGHVWKV Based on the above, I would call the above Glaske16 & Dulcita genes phosphoesterases. Or could I be missing something? Thanks! Fred 365Kb

Link to this post | posted 01 Jun, 2023 16:14

fbaliraine

My DNA Master was working well till this morning when I tried updating it. It couldn't, even after re-starting the computer. I asked our IT to uninstall and Re-install it (I had issues with DNA Master last week and re-installing worked!). But when I got in to set the preferences, I noticed that several options for the suggested preferences were missing, including the options to insert template into Notes, Direct connections to servers to obtain Glimmer and GeneMark data/Gene Prediction Server Location, Secure Connections, and the option to set the Shine Dalgarno Scoring. When I tried using it to check RBS score, I can no longer see the z values, etc., all I can see is the "SD Score" & "space" (See attached). Is this the new normal, or is something happening with DNA Master? Also, looks like there could be problems with the server, as IT gets and the following "Error connecting to cobamide2.bio.pitt.edu" Fred Edited 01 Jun, 2023 20:30 35Kb