SEA-PHAGES | All posts created by kbutela

Link to this post \| posted 02 Jul, 2018 20:37
kbutela	NEHalo gp3 (http://phagesdb.org/genes/NEHALO_DRAFT_3/) currently Pham 37118 ) is called in several phages as "terminase, small subunit" or "tail protein" in four other phages. The sole HHPred match at 94% probability is to PF05119.11 Terminase_4, phage terminase, small subunit but is not linked to a crystal structure in the PDB. This match has an E-value of 0.17 with coverage of 47%. All other matches show low probability scores of 40% or below. COILS did not reveal the coil-coiled domain that is typically found in many small subunit terminase sequences in gp3. I ran the sequence through Phyre2 (output attached) and got low confidence matches to the regulatory domain of mycobacterial adenylyl cyclase rv1264 (80; 12% id) and a phage tail protein (40.6, 22% id). No other evidence of the call for terminase, small subunit was found other than Blast matches to previously annotated phages and that one HHPred hit with low e-value. Should this call of "terminase, small subunit" be changed to NKF? Gp10 is annotated as "terminase, large subunit" and has well supported HHPred matches to other large terminases. There are a few NKF genes located in between the putative "terminase, small subunit" gp3 and gp10 from a synteny perspective. Edited 02 Jul, 2018 20:39 282Kb

Link to this post | posted 02 Jul, 2018 20:37

NEHalo gp3 (http://phagesdb.org/genes/NEHALO_DRAFT_3/) currently Pham 37118 ) is called in several phages as "terminase, small subunit" or "tail protein" in four other phages.

The sole HHPred match at 94% probability is to PF05119.11 Terminase_4, phage terminase, small subunit but is not linked to a crystal structure in the PDB. This match has an E-value of 0.17 with coverage of 47%. All other matches show low probability scores of 40% or below.

COILS did not reveal the coil-coiled domain that is typically found in many small subunit terminase sequences in gp3. I ran the sequence through Phyre2 (output attached) and got low confidence matches to the regulatory domain of mycobacterial adenylyl cyclase rv1264 (80; 12% id) and a phage tail protein (40.6, 22% id). No other evidence of the call for terminase, small subunit was found other than Blast matches to previously annotated phages and that one HHPred hit with low e-value.

Should this call of "terminase, small subunit" be changed to NKF? Gp10 is annotated as "terminase, large subunit" and has well supported HHPred matches to other large terminases. There are a few NKF genes located in between the putative "terminase, small subunit" gp3 and gp10 from a synteny perspective.

Edited 02 Jul, 2018 20:39

Posted in: Functional Annotation → Cluster A1 terminase, small subunit

Link to this post \| posted 10 May, 2018 14:34
kbutela	I've seen this happen a few times. I have my students set up a DNA Master Archive folder somewhere on their Mac, then I have them set this folder to default in DNA Master Preferences. I discourage my students from using the WINE "DNA Master Archive" folder since sometimes files in there seem to get lost or corrupted more easily. Are the students opening files from the DNA Master "open" menu? In my experience, there is usually a brief hang time for opening files. If the program still isn't opening files, then I have the students uninstall and reinstall the program which seems to take care of the problem. Randall DeJong This will hopefully be a silly, easy-to-answer question! We don't typically use DNA Master for Mac in our course, but we have some students who wish to continue unfinished annotations into the summer. They have downloaded and installed the Wine Bottle version posted by Baylor (thanks SEA colleagues!), and it appears to run fine. However, some run into trouble in that they cannot open files - they just hang. My suspicion is that the files need to be in the DNA Master archives folder, only those files aren't 'seen' when in the Mac Finder. Is my suspicion correct or is something else? Any solutions? Thanks!

Link to this post | posted 10 May, 2018 14:34

kbutela

I've seen this happen a few times. I have my students set up a DNA Master Archive folder somewhere on their Mac, then I have them set this folder to default in DNA Master Preferences. I discourage my students from using the WINE "DNA Master Archive" folder since sometimes files in there seem to get lost or corrupted more easily. Are the students opening files from the DNA Master "open" menu? In my experience, there is usually a brief hang time for opening files. If the program still isn't opening files, then I have the students uninstall and reinstall the program which seems to take care of the problem.

Randall DeJong
This will hopefully be a silly, easy-to-answer question!
We don't typically use DNA Master for Mac in our course, but we have some students who wish to continue unfinished annotations into the summer. They have downloaded and installed the Wine Bottle version posted by Baylor (thanks SEA colleagues!), and it appears to run fine. However, some run into trouble in that they cannot open files - they just hang. My suspicion is that the files need to be in the DNA Master archives folder, only those files aren't 'seen' when in the Mac Finder. Is my suspicion correct or is something else? Any solutions?

Thanks!

Posted in: DNA Master → Running DNA Master on a Mac using Wine

Link to this post \| posted 08 May, 2018 19:12
kbutela	Hi Evan- This has happened to me on occasion while running the WINE version of DNA Master, most often if I have two files open at the same time and try to do a Frames view of both files. I'm guessing that some sort of memory issue may be at play here. Does this happen every time you try to work with this phage, and when this phage is the only .dnam5 file open in DNA Master? If you can send the file to me I can try to duplicate the error to see what's going on. Best, Kristen Evan Merkhofer Second error message.

Link to this post | posted 08 May, 2018 19:12

kbutela

Hi Evan-

This has happened to me on occasion while running the WINE version of DNA Master, most often if I have two files open at the same time and try to do a Frames view of both files. I'm guessing that some sort of memory issue may be at play here. Does this happen every time you try to work with this phage, and when this phage is the only .dnam5 file open in DNA Master? If you can send the file to me I can try to duplicate the error to see what's going on.

Best,

Kristen

Evan Merkhofer
Second error message.

Posted in: DNA Master → Running DNA Master on a Mac using Wine

Link to this post \| posted 17 Aug, 2017 02:53
kbutela	Cluster B2s lack lysin B. I thought there was pretty good evidence of pham 609 being annotated as lysin B based on HHPred and TMM data. In this 2016 paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912749/) they have Numberten and Pops (both B1s) containing both lysin A and lysin B.

Posted in: Cluster B Annotation Tips → Lysin B

Link to this post \| posted 17 Aug, 2017 02:49
kbutela	In reviewing a few Cluster B1 phages, I am seeing some members of Pham (Webphamerator) 7880 (see Vivaldi gp64 for an example) as recombination directionality factor/RDF/RDF protein. I thought that the Cluster B phages are not temperate and aren't known to contain integrase genes. Why would a recombination directionality factor be present in a non-temperate genome, and apparently conserved among several other related phages? Is this a mis-annotation or am I not understanding something given that I'm writing this a little late at night? The only HHPred results don't meet the threshold for acceptance but they are close (high 80s%) to zinc finger domains. No conserved domains were identified. 114Kb

Link to this post | posted 17 Aug, 2017 02:49

kbutela

In reviewing a few Cluster B1 phages, I am seeing some members of Pham (Webphamerator) 7880 (see Vivaldi gp64 for an example) as recombination directionality factor/RDF/RDF protein. I thought that the Cluster B phages are not temperate and aren't known to contain integrase genes. Why would a recombination directionality factor be present in a non-temperate genome, and apparently conserved among several other related phages? Is this a mis-annotation or am I not understanding something given that I'm writing this a little late at night? The only HHPred results don't meet the threshold for acceptance but they are close (high 80s%) to zinc finger domains. No conserved domains were identified.

Posted in: Functional Annotation → Recombination directionality factor in Cluster B1s

Link to this post \| posted 11 Aug, 2017 21:39
kbutela	The documentation in the COILS2 help guide suggested a cutoff of 0.5 for a residue to be a part of a coiled coil motif. I may be a bit conservative in my interpretations, but I'm going to stick with the 0.5 cutoff for now. I attached a screenshot of the results I got when I ran Baka_5 through COILS2 using default parameters. 13Kb

Posted in: Functional Annotation → cluster J terminases

Link to this post \| posted 11 Aug, 2017 21:03
kbutela	Presence of a coiled coil motif. I ran Baka_5 as an example and very clearly saw the CCM Jackie identified (prob. was at least 0.6). Per our discussion on the Cluster A4 terminase genes from yesterday, I ran Blackmoor_2 through COILS2. Although this gene has HHPred hits (87% probability, 53% coverage) to a phage terminase, the probability of a CCM is only about 0.15. I played around with the scoring algorithms recommended in the manual and never got anything better. TinaFeyge_2 is annotated as a terminase, small subunit, but I also didn't get a CCM motif for this gene on COILS2. The evidence is close, but I don't think it meets the threshold to definitively call this gene in the A4s as a small terminase.

Link to this post | posted 11 Aug, 2017 21:03

kbutela

Presence of a coiled coil motif. I ran Baka_5 as an example and very clearly saw the CCM Jackie identified (prob. was at least 0.6). Per our discussion on the Cluster A4 terminase genes from yesterday, I ran Blackmoor_2 through COILS2. Although this gene has HHPred hits (87% probability, 53% coverage) to a phage terminase, the probability of a CCM is only about 0.15. I played around with the scoring algorithms recommended in the manual and never got anything better. TinaFeyge_2 is annotated as a terminase, small subunit, but I also didn't get a CCM motif for this gene on COILS2. The evidence is close, but I don't think it meets the threshold to definitively call this gene in the A4s as a small terminase.

Posted in: Functional Annotation → cluster J terminases

Link to this post \| posted 08 Aug, 2017 20:02
kbutela	Pham 5142 (as of 8/8/17) corresponds to several members in Cluster B1, some of which are annotated as PLA2, PLA2-like domain, and PLA2-like domain protein, with PLA2 standing for phospholipase A2. According to this paper, some bacteria contain phospholipase A2, and a quick Blast search shows phospholipase A2 in Mycobacterium tuberculosis in two forms, one 512 aa long and the other designated as "membrane associated" at 111 aa long. Most members of Pham 5142 are relatively small, in the range of 40-50 aa. This gene (Virapocalypse gp73) does not match to any "PLA2" designated proteins in HHPred (attached) or in NCBI Blast other than mycobacteriophages. No conserved domains were found. I pulled the M. tuberculosis phospholipase A protein sequence (512 aa) and the one designated as "Membrane associated" as well as a sequence pulled from the crystal structure of phospholipase A from Streptomyces (1FAZ) and ran them through MUSCLE, Clustal Omega, and T-Coffee; all three give pretty lousy alignments with gp73 of Virapocalypse. I am not convinced this gene contains a PLA2 domain, but I'm not sure where the call of PLA2-like domain originated since the functional source for Virapocalpyse is ShiVal gp71 (older, annotated in 2011). Thoughts? 113Kb

Link to this post | posted 08 Aug, 2017 20:02

kbutela

Pham 5142 (as of 8/8/17) corresponds to several members in Cluster B1, some of which are annotated as PLA2, PLA2-like domain, and PLA2-like domain protein, with PLA2 standing for phospholipase A2. According to this paper, some bacteria contain phospholipase A2, and a quick Blast search shows phospholipase A2 in Mycobacterium tuberculosis in two forms, one 512 aa long and the other designated as "membrane associated" at 111 aa long. Most members of Pham 5142 are relatively small, in the range of 40-50 aa.

This gene (Virapocalypse gp73) does not match to any "PLA2" designated proteins in HHPred (attached) or in NCBI Blast other than mycobacteriophages. No conserved domains were found. I pulled the M. tuberculosis phospholipase A protein sequence (512 aa) and the one designated as "Membrane associated" as well as a sequence pulled from the crystal structure of phospholipase A from Streptomyces (1FAZ) and ran them through MUSCLE, Clustal Omega, and T-Coffee; all three give pretty lousy alignments with gp73 of Virapocalypse. I am not convinced this gene contains a PLA2 domain, but I'm not sure where the call of PLA2-like domain originated since the functional source for Virapocalpyse is ShiVal gp71 (older, annotated in 2011). Thoughts?

Posted in: Functional Annotation → PLA2-like domain in Cluster B1s

Link to this post \| posted 26 Apr, 2017 22:29
kbutela	I've been BLASTing just fine over the last few days. My DNA Master version is 5.23.1, build 2492, 29 January 2017. A lot of people, including myself, seemed to have trouble earlier in the semester, and I seem to recall that the group conclusion (maybe on another post?) was that the NCBI servers were getting too many simultaneous requests from the same place/school, so Blast was kicking people out, resulting in DNA Master throwing the error.

Link to this post | posted 26 Apr, 2017 22:29

kbutela

I've been BLASTing just fine over the last few days. My DNA Master version is 5.23.1, build 2492, 29 January 2017. A lot of people, including myself, seemed to have trouble earlier in the semester, and I seem to recall that the group conclusion (maybe on another post?) was that the NCBI servers were getting too many simultaneous requests from the same place/school, so Blast was kicking people out, resulting in DNA Master throwing the error.

Posted in: DNA Master → Running DNA Master on a Mac using Wine

Link to this post \| posted 02 Feb, 2017 22:21
kbutela	I'm running WINE DNA Master (29 Jan 2017 update) on a Mac OSX El Capitan 10.11.6, XQuartz version 2.7.6. I just ran autoannotation on some test files, and Glimmer appears to be working. Occasionally, I have encountered the Glimmer failure error, and just redoing the autoannotation later seemed to fix the problem. Keith's suggestion that the Glimmer servers may be getting clogged or rejecting multiple queries from the same IP domain in a limited amount of time may be a good explanation of what's going on.

Link to this post | posted 02 Feb, 2017 22:21

kbutela

I'm running WINE DNA Master (29 Jan 2017 update) on a Mac OSX El Capitan 10.11.6, XQuartz version 2.7.6.

I just ran autoannotation on some test files, and Glimmer appears to be working. Occasionally, I have encountered the Glimmer failure error, and just redoing the autoannotation later seemed to fix the problem. Keith's suggestion that the Glimmer servers may be getting clogged or rejecting multiple queries from the same IP domain in a limited amount of time may be a good explanation of what's going on.

Posted in: DNA Master → Glimmer Failure on Auto Annotation

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