Link to this post | posted 02 Jun, 2020 21:29

GregFrederick@letu.edu

Hi all. I just ran into a DNA Master error that I've not seen in the five plus years we have been in the program. I searched the forums, but did not find a solution. PROBLEM: A newly saved DNAM5 file when reopened did not list the features in the feature table. The documentation appeared correct. I could see all the features listed correctly with coordinates and functions in the documentation. I went through the process described in the Annotation Guide to "Parse the Documentation". That is linked here: https://seaphagesbioinformatics.helpdocsonline.com/article-93 However, this did not resolve the problem on the original DNAM5 file. SOLUTION: I watched this video where Welkin Pope describes how to repair a corrupted DNAM5 file. https://vimeo.com/277487017 I went through all the steps that she included. When I opened the repacked file, I still could not see any features listed in the feature list. Then I went back to the documentation tab and could see the features listed there. One more reparse recreated the features in the feature tab. Now everything has been restored EXCEPT that the Blast Data appears to have been lost. There were two blast file in the directory. (QBlastHit0.MB and QBlastHSP0.MB) However, that data was apparently not pulled in on the repackaging. I'll rerun the Blast analysis tonight. FINAL COMMENT: The one step I needed to add beyond Welkin's video was to reprase the documentation after the repackaged file was created. Good luck! Edited 02 Jun, 2020 22:07

Link to this post | posted 14 May, 2020 18:12

GregFrederick@letu.edu

Debbie. I just want to make certain I am making calls correctly. The responses above left me a bit uncertain. I'm going to ask a couple of follow-up questions. Admittedly I have not looked into Jamie's genome closely. But the responses above are making us question a call we made on Phage Heath gp71. Let's look at this from general gene calls initially, if I may. 1. If I were looking at HHPred results for any gene and saw 10 or more hits, all with similar protein functions, covering nearly half the protein with a probability of over 98% and e-values of 10^-6 or better I would lean toward calling the HHPred identified function. Wouldn't this be strong enough supporting evidence for at least considering? 2. If the same gene had a first HHPred hit that is the second HHPred hit for the Official Function List reference gene, I would again feel more confident with the function call, even if no one else in the SEA had noticed it previously. Is this consideration wrong? 3. If that first HHPred hit is a protein from Mycobacterium tuberculosis, I would tend to feel even more confident the call is correct, even though no other PhagesDB Blast hits to the protein have ID'd this function. Is this consideration wrong? At that point, I would likely assign function based upon HHPred analysis results alone. Again, I don't know all the details in Jamie's phage and how strong his hits are. We were about to switch the call to "Hypothetical Protein" based upon the response above. But my gut tells me I need to ask these questions to make certain. I may have been doing this wrong in the past. IDK. The HHPred output link to (Heath_gp71) the gene we are now questioning is here: https://toolkit.tuebingen.mpg.de/jobs/Heath_gp71 That linked output will likely go away in 20-30 days. So I will attach the results in a pdf for future reference. The HHPred hit 6DVC_F (our first hit) is the second hit for the reference phage gene (Nerujay_52) for RNA Polymerase Sigma Factor. We thought this would be enough supporting evidence. But we don't want to call it if it should not be. We, also don't want to "band wagon" and call it like everyone else, if this is sufficient supporting evidence to make a functional call. Thanks for any additional clarification you can provide on using HHPred hits alone for function assignment. Greg 898Kb

Link to this post | posted 12 May, 2020 16:01

GregFrederick@letu.edu

I had not realized there was a problem with MacOS10 Catalina until COVID caused us to disperse. Students with Macs had been relying on the PCs in the university computer labs. Once they did not have access, we had to scramble. The easiest solution I could find (I have not used a Mac in years other than to keep my wife's iMac running), was to give students instructions on how to run "Boot Camp" on their Macs and install Windows 10 as a second boot environment. MS Win10 is free to our students through the university. Every student that needed to install DNA Master on their Mac with MacOS Catalina, was able to do so. Instructions on installing MS Win10 with Boot Camp on a Mac are available here: https://support.apple.com/boot-camp Hopefully this is helpful. Greg

Link to this post | posted 31 Mar, 2020 21:57

GregFrederick@letu.edu

Hi all. The great COVID-19-diaspora of 2020 has revealed some student-errors. Apparently a number were relying solely on the computer lab PCs and never got DNA Master installed on their MacBooks. I helped those that came to me. But I have 3-4 who don't have it installed. QUESTION: Has anyone found a way to install the Wine DNA Master package on a Mac running the Catalina 10.15.x Mac OS? The DNA Master software acts like it is installing. An icon displays on the desktop. But once clicked, the DNA Master icon just bounces and does nothing more. Working remotely is making it difficult to troubleshoot. So I'm hoping you all have suggestions. Thanks. Greg

Link to this post | posted 28 Feb, 2020 15:51

GregFrederick@letu.edu

OK "Smarty Pants". You were right. There are indeed two "Ns" at the end of the sequence. However, we do not know how to remove them (or how they got inserted). For completion sake on this thread, could you please describe the process for removing them? (My student has deleted them, changed the feature list, posted and saved. But the "Ns" return in the saved .DNAM5 file once reopened. So a functional protocol would be very helpful and might help others down the road. By the way, the student thinks you must be a wizard to have known about the Ns! ) Edited 28 Feb, 2020 15:52

Link to this post | posted 27 Feb, 2020 23:06

GregFrederick@letu.edu

We just attempted to merge the DNAM5 files from 4 different students. Apparently, 2 somehow change the DNA sequence length. Two genomes are 2bp longer than the original. They have completed the annotation of their section of the genome. But because of the different lengths, we cannot merge files. QUESTIONS: 1. Is there a way to align the two DNA sequence files to determine where the nucleotides have been added or deleted? 2. If not, what is the easiest solution. OUR CURRENT SOLUTION: The students can see where the genome length actually changed in their various file reiteration and save. They have gone back to the file with the correct genome length and are copying notes from the version with the wrong length. They are also readjusting start codons for each gene. Is there an easier way to do this? Obviously had we noticed the 2bp length change when it happened would could have eliminated a lot of work by going one file iteration back instead of about 20…. {Frowny face emoji} Edited 27 Feb, 2020 23:07

Link to this post | posted 23 Jan, 2020 21:17
GregFrederick@letu.edu	The server (or our network) apparently came back online shortly after class ended at 10:30am CST. It may have been our network. Who knows. Since we are still in "training mode" we just had the students huddle around those computers which would update. Thanks for the response. gf Edited 23 Jan, 2020 21:17

Link to this post | posted 23 Jan, 2020 15:13
GregFrederick@letu.edu	Is anyone else having difficulty updating DNA Master this morning? It's 9:08AM CST and over half our lab computers (all of which have updated fine previously) are not able to progress beyond 67% in the update. Anybody else?

Link to this post | posted 12 Apr, 2019 00:31

GregFrederick@letu.edu

All the hits ID'd as "Teriminase Large Subunit" are in the N-terminus of the proteins. After reading this: Structure of the large terminase from a hyperthermophilic virus reveals a unique mechanism for oligomerization and ATP hydrolysis. Xu, R.G., Jenkins, H.T., Antson, A.A., Greive, S.J. (2017) Nucleic Acids Res. 45: 13029-13042 PubMed: 29069443 Search on PubMed DOI: 10.1093/nar/gkx947 Primary Citation of Related Structures: 5OEE, 5OEB, 5OEA, 5OE9 PubMed Abstract: The crystal structure of the large terminase from the Geobacillus stearothermophilus bacteriophage D6E shows a unique relative orientation of the N-terminal adenosine triphosphatase (ATPase) and C-terminal nuclease domains. This monomeric 'initiation' state with the two domains 'locked' together is stabilized via a conserved C-terminal arm, which may interact with the portal protein during motor assembly, as predicted for several bacteriophages. . . . . It seems we are hitting the "ATP Binding Cassette" region. Some of the lower level HHPred hits indicate weak hits to ABCs or ATPases in other proteins. Edited 12 Apr, 2019 00:34

Link to this post | posted 12 Apr, 2019 00:04

GregFrederick@letu.edu

Thanks Debbie, I don't think you need to investigate further. It seems we had a student-faculty communication breakdown. They originally reported an HHPred report with a 94% coverage. Their calculations were based on 94% of the Phalm gp68 protein aligning with the HHPred target hit. My calculations (above) were the % coverage by the Phalm gp68 protein of the HHPred hit. The Phalm gp68 only covers 18-28% of the hits above. After we discussed, I realized that even though we have a P value of 90-92 in each case, the gp68 protein is only 84aa in length. It is not large enough to be a "terminase large subunit" protein. It probably does hit one domain. But,from the HHPred results, it is not obvious which domain that it might be. We'll try to dig a little deeper. Thanks.